Background and objectives:Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) in humans. Poor treatment responses are a threat to global TB control, as such, understanding contributing factors to poor responses is important. We proposed that antibiotic tolerance could contribute to delayed culture conversion (recalcitrant TB), and resistance amplification in patients during TB treatment. We thus ventured to investigate the role of drug tolerance in delayed culture conversion and resistance amplification in TB patients. Methods:We collected serial Mtb isolates from patients with (i) drug-susceptible TB who remained culture positive for up to 6 years (i.e. recalcitrant TB), and (ii) multidrug-resistant TB (MDR-TB) where resistance amplified during treatment. We measured tolerance to rifampicin in drug-susceptible TB strains and tolerance to moxifloxacin in MDR-TB strains using a real-time time-kill assay. Results and discussion:Rifampicin tolerance evolved within-host, increasing up to and ∼1.5-fold, however, there was no apparent contribution of rifampicin tolerance to delayed culture conversion. Tolerance to moxifloxacin in MDR-TB patients appeared negatively associated with resistance amplification and consistently decreased over time in patients. Conclusion:Our findings confirm that antibiotic tolerance evolves in Mtb within patients over time during treatment. However, there was no evidence that this tolerance influences treatment responses, calling for further investigation of contributors to adverse treatment responses and their mitigation.

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.

The Mycobacterium tuberculosis complex (MTBC) includes ten human-adapted lineages with varying geography and pathogenicity. Lineage 1 (L1) shows low virulence while Lineage 2 (L2) is hyper-virulent, more transmissible, and associated with drug-resistance. We performed comparative analyses integrating whole-genome sequencing with transcriptomic and proteomic profiling of L1 and L2 clinical strains under two in vitro growth conditions. Transcript-protein correlations varied by strain and gene category, suggesting lineage-specific post-translational regulation. Expression differences scaled with phylogenetic distance, one in three SNPs affected gene expression. A new transcriptional regulatory model identified master transcription factors, linked to the sigma factor network, whose targets were differentially expressed between L1 and L2. For instance, DosR regulon proteins had higher basal levels and exhibited a stronger nitric oxide response in L2. Time-course experiments involving LysG (Rv1985c) induction and wild-type H37Rv under hypoxia and subsequent reaeration confirmed that LysG contributes to reduced metabolic activity, thereby promoting increased tolerance to the novel tuberculosis drug bedaquiline in L2 strains relative to L1. Overall, our findings show how limited genetic variation in the MTBC can yield major phenotypic differences through differential regulation of key transcriptional networks.

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.

In vitro antibiotic testing is important for guiding therapy and drug development. Current methods are focused on growth inhibition in bulk bacterial populations but often fail to accurately predict treatment responses. Here we introduce Antimicrobial Single-Cell Testing (ASCT), a large-scale live-cell imaging approach that quantifies bacterial killing in real time at single-cell resolution. By tracking over 140 million mycobacteria and analysing ~20,000 time–kill curves, we identify key determinants of antibiotic killing and its clinical relevance. For Mycobacterium tuberculosis, we found that drug-specific killing dynamics in starved bacteria, rather than growth inhibition or killing of growing cells, predict regimen efficacy in mice and humans. Extending this approach to Mycobacterium abscessus and comparing 405 bacterial strains, we show that antibiotic killing is also a genetically encoded bacterial trait (drug tolerance). We demonstrate that tolerance patterns cluster by antibiotic targets, identify a phage protein that modulates antibiotic killing, and show that strain-specific killing dynamics are associated with individual patient outcomes independent of drug resistance. Together, these findings establish a framework that reveals how drug properties and bacterial diversity shape treatment responses, offering a path to more effective and personalized therapies. Via high-throughput imaging and tracking over 140 million single mycobacteria, the authors show that drug- and strain-specific killing predict treatment outcomes, with potential to improve drug development and personalized therapy.

The members of the Mycobacterium tuberculosis complex (MTBC) causing human tuberculosis comprise 10 phylogenetic lineages that differ in their geographical distribution. The human consequences of this phylogenetic diversity remain poorly understood. Here, we assessed the phenotypic properties at the host-pathogen interface of 14 clinical strains representing five major MTBC lineages. Using a human in vitro granuloma model combined with bacterial load assessment, microscopy, flow cytometry, and multiplexed-bead arrays, we observed considerable intra-lineage diversity. Yet, modern lineages were overall associated with increased growth rate and more pronounced granulomatous responses. MTBC lineages exhibited distinct propensities to accumulate triglyceride lipid droplets—a phenotype associated with dormancy—that was particularly pronounced in lineage 2 and reduced in lineage 3 strains. The most favorable granuloma responses were associated with strong CD4 and CD8 T cell activation as well as inflammatory responses mediated by CXCL9, granzyme B, and TNF. Both of which showed consistent negative correlation with bacterial proliferation across genetically distant MTBC strains of different lineages. Taken together, our data indicate that different virulence strategies and protective immune traits associate with MTBC genetic diversity at lineage and strain level.

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.

INTRODUCTION:Resistance to bedaquiline - a novel, promising medication against tuberculosis (TB), is already emerging, and uncertainties regarding the role of the different resistance-conferring mutations complicate the development of molecular diagnostic tools for detecting resistance. Mutations in the three genes atpE, pepQ, and Rv0678 have been associated with increased minimum inhibitory concentrations (MICs) to bedaquiline in Mycobacterium tuberculosis (Mtb). Here, we studied the effect of known and novel mutations in these genes on the phenotypic susceptibility to bedaquiline in Mtb isolates from patients with drug-resistant TB in the country of Georgia. METHODS:We used retrospective Mtb isolates (2011-2019) with whole-genome sequencing data, and prospectively collected diagnostic isolates with phenotypic resistance (2019-2022) to bedaquiline at the Georgian National Reference Laboratory. We determined bedaquiline MIC values using the SensititreTM MYCOTB MIC plate. MIC of 0.12 μg/mL was defined as borderline and MIC ≥ 0.25 μg/mL as a resistant isolate. A phylogeny was inferred to assess the likely role of the identified variants in bedaquiline resistance, while taking into consideration population structure of the strains analyzed. RESULTS:We analyzed a total of 69 Mtb isolates and identified 61 mutations across the three target genes. Seventeen (27.8%) of these variants were associated with borderline (0.12 μg/mL) or resistant (≥0.25 μg/mL) MICs to bedaquiline. In addition to six previously described bedaquiline resistance-conferring mutations in atpE and Rv0678, we identified two novel variants in Rv0678 (Leu95Ser and Ile108fs) likely involved in bedaquiline resistance. We found a Tyr92Cys mutation in Rv0678 in two epidemiologically linked isolates, which likely emerged as a consequence of previous exposure to clofazimine. CONCLUSION:Consistent with previous reports, our study confirms that mutations in Rv0678 are the most frequent cause of bedaquiline resistance in Georgia, in addition to an increasing clinical relevance of mutations in atpE, while the role of pepQ mutations remains to be defined.

Under stress, bacteria display selective growth propensities on solid and liquid media. For Mycobacterium tuberculosis (Mtb), differentially culturable bacteria have been found in tuberculosis (TB) patient sputa. We hypothesized that antibiotic treatment can induce selective culturability in Mtb. We investigated the effect of exposure to TB drugs on Mtb culturability using clinical samples from an ongoing TB patient cohort and by conducting several in vitro experiments with a diverse set of Mtb strains. In patients, serial sputa were more likely to generate Mtb-positive cultures in liquid as opposed to solid medium, with this liquid culture bias extending up to 5 months post diagnosis. Experimentally, there was a disparity between bacterial time to positivity (TTP) and colony forming units (CFUs) when Mtb was exposed to isoniazid (INH) and rifampicin (RIF) alone or in combination. Cultures recovered from RIF treatment yielded more CFUs on agar plates, but INH-treated cultures had a faster TTP in liquid. Follow up experiments using a fluorescently labelled laboratory strain of Mtb revealed that CFUs of INH-treated bacteria were too low, thus over-estimating the killing effect of the treatment. Here we provide evidence that drug exposure affects culturability on solid medium in diverse Mtb strains, which has implications for treatment monitoring and drug-pathogen interaction studies.

Studies in model organisms show that mutations conferring resistance to different antibiotics can interact epistatically, but the biological and epidemiological consequences of such interactions are unknown. Here we show that in Mycobacterium tuberculosis (Mtb) , positive sign epistasis between RpoB and GyrA mutations causing resistance to rifampicin and fluoroquinolone, respectively, can lead to double-resistant strains with high in vitro fitness. Two of these RpoB-GyrA mutation combinations account for 53% in a global collection of highly drug-resistant Mtb clinical isolates, compared to <0.7% for RpoB-GyrA combinations with low in vitro fitness. Moreover, the two high-fitness RpoB-GyrA combinations are associated with a more benign and idiosyncratic proteome perturbation compared to low-fitness combinations. Our findings highlight the relevance of epistasis for the emergence and spread of antimicrobial resistance. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 883285 Swiss National Science Foundation, https://ror.org/00yjd3n13, CRSII5_213514, 10000213, 10001893

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.

Antibiotic development and treatment focus on bacterial growth inhibition, often with limited success. Here, we introduce Antimicrobial Single-Cell Testing (ASCT), an advanced imaging strategy to assess bacterial killing in real-time. By tracking 140 million bacteria and generating over 20,000 in vitro time-kill curves, we can predict Mycobacterium tuberculosis treatment outcomes in mice and humans and link strain-specific survival (drug tolerance) in Mycobacterium abscessus to clinical responses. Using ASCT, we reveal drug tolerance as a distinct genetically encoded bacterial trait conserved across drugs with similar targets and, via genome-wide associations, uncover molecular mechanisms that govern bacterial killing. This study establishes the technical framework and in vivo validation for large-scale bacterial killing assessments to advance our understanding of bacterial survival and enhance antibiotic development and clinical decision-making. ### Competing Interest Statement The authors have declared no competing interest.