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Abstract 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.

Geographical hotspots with high frequency of multi-drug resistant tuberculosis (MDR-TB) have been observed in several locations, such as the country of Georgia. Here, the authors analyse genomic sequences from tuberculosis bacteria isolated from Georgia to show that the transmission fitness of MDR-TB strains is heterogeneous, and highly drug-resistant and transmissible isolates contribute to the emergence and maintenance of MDR-TB hotspots.

An outbreak of the multidrug-resistant Mycobacterium tuberculosis lineage W148 has spread widely across Russia, Central Asia and Europe. Here, the authors use whole genome sequences of ~700 isolates of this lineage collected over ~20 years to analyze its spread, evolution of drug resistance, and impact of compensatory mutations.

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.

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.

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.

Introduction Mycobacterial Interspersed Repetitive Units–Variable Tandem Repeats (MIRU-VNTR) typing has been widely used for molecular epidemiological studies of tuberculosis (TB). However, genotyping tools for Mycobacterium tuberculosis (Mtb) may be limiting in some settings due to high cost and workload. In this study developed a customized stepwise MIRU-VNTR typing that prioritizes high discriminatory loci and validated this method using penitentiary system cohort in the country of Georgia. Methods We used a previously generated MIRU-VNTR dataset from recurrent TB cases (32 cases) in Georgia and a new dataset of TB cases from the penitentiary system (102 cases) recruited from 2014 to 2015. A Hunter-Gaston Discriminatory Index (HGDI) was calculated utilizing a 24 standard loci panel, to select high discriminatory power loci, subsequently defined as the customized Georgia-specific set of loci for initial typing. The remaining loci were scored and hierarchically grouped for second and third step typing of the cohort. We then compared the processing time and costs of the customized stepwise method to the standard 24-loci method. Results For the customized Georgia-specific set that was used for initial typing, 10 loci were selected with a minimum value of 0.32 to the highest HGDI score locus. Customized 10 loci (step 1) typing of 102 Mtb patient isolates revealed 35.7% clustered cases. This proportion was reduced to 19.5% after hierarchical application of 2nd and 3rd step typing with the corresponding groups of loci. Our customized stepwise MIRU-VNTR genotyping approach reduced the quantity of samples to be typed and therefore overall processing time and costs by 42.6% each. Conclusion Our study shows that our customized stepwise MIRU-VNTR typing approach is a valid alternative of standard MIRI-VNTR typing panels for molecular epidemiological investigation in Georgia that saves time, workload and costs. Similar approaches could be developed for other settings.

Summary: Background: South Africa has a high burden of rifampicin-resistant tuberculosis (including multidrug-resistant [MDR] tuberculosis), with increasing rifampicin-monoresistant (RMR) tuberculosis over time. Resistance acquisition during first-line tuberculosis treatment could be a key contributor to this burden, and HIV might increase the risk of acquiring rifampicin resistance. We assessed whether HIV during previous treatment was associated with RMR tuberculosis and resistance acquisition among a retrospective cohort of patients with MDR or rifampicin-resistant tuberculosis. Methods: In this retrospective cohort study, we included all patients routinely diagnosed with MDR or rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa, between Jan 1, 2008, and Dec 31, 2017. Patient-level data were obtained from a prospective database, complemented by data on previous tuberculosis treatment and HIV from a provincial health data exchange. Stored MDR or rifampicin-resistant tuberculosis isolates from patients underwent whole-genome sequencing (WGS). WGS data were used to infer resistance acquisition versus transmission, by identifying genomically unique isolates (single nucleotide polymorphism threshold of five). Logistic regression analyses were used to assess factors associated with RMR tuberculosis and genomic uniqueness. Findings: The cohort included 2041 patients diagnosed with MDR or rifampicin-resistant tuberculosis between Jan 1, 2008, and Dec 31, 2017; of those, 463 (22·7%) with RMR tuberculosis and 1354 (66·3%) with previous tuberculosis treatment. In previously treated patients, HIV positivity during previous tuberculosis treatment versus HIV negativity (adjusted odds ratio [OR] 2·07, 95% CI 1·35–3·18), and three or more previous tuberculosis treatment episodes versus one (1·96, 1·21–3·17) were associated with RMR tuberculosis. WGS data showing MDR or rifampicin-resistant tuberculosis were available for 1169 patients; 360 (30·8%) isolates were identified as unique. In previously treated patients, RMR tuberculosis versus MDR tuberculosis (adjusted OR 4·96, 3·40–7·23), HIV positivity during previous tuberculosis treatment (1·71, 1·03–2·84), and diagnosis in 2013–17 (1·42, 1·02–1·99) versus 2008–12, were associated with uniqueness. In previously treated patients with RMR tuberculosis, HIV positivity during previous treatment (adjusted OR 5·13, 1·61–16·32) was associated with uniqueness as was female sex (2·50 [1·18–5·26]). Interpretation: These data suggest that HIV contributes to rifampicin-resistance acquisition during first-line tuberculosis treatment and that this might be driving increasing RMR tuberculosis over time. Large-scale prospective cohort studies are required to further quantify this risk. Funding: Swiss National Science Foundation, South African National Research Foundation, and Wellcome Trust.

Summary: Background: Drug resistance threatens global tuberculosis control. We aimed to examine mortality in patients with tuberculosis from high-burden countries, according to concordance or discordance of results from drug susceptibility testing done locally and whole-genome sequencing (WGS). Methods: In this multicentre cohort study, we collected pulmonary Mycobacterium tuberculosis isolates and clinical data from individuals with tuberculosis from antiretroviral therapy programmes and tuberculosis clinics in Côte d'Ivoire, Democratic Republic of the Congo, Kenya, Nigeria, Peru, South Africa, and Thailand, stratified by HIV status and drug resistance. Sites tested drug susceptibility using routinely available methods. WGS was done on Illumina HiSeq 2500 in the USA and Switzerland, and TBprofiler was used to analyse the genomes. We included individuals aged 16 years or older with pulmonary tuberculosis (bacteriologically confirmed or clinically diagnosed). We analysed mortality in multivariable logistic regression models adjusted for sex, age, HIV status, history of tuberculosis, and sputum positivity. Findings: Between Sept 1, 2014, and July 4, 2016, of 634 patients included in our previous analysis, we included 582 patients with tuberculosis (median age 33 years [IQR 27–43], 225 [39%] women, and 247 [42%] HIV-positive). Based on WGS, 339 (58%) isolates were pan-susceptible, 35 (6%) monoresistant, 146 (25%) multidrug-resistant, and 24 (4%) pre-extensively drug-resistant (pre-XDR) or XDR. The analysis of mortality was based on 530 patients; 63 (12%) died and 77 (15%) patients received inappropriate treatment. Mortality ranged from 6% (18 of 310) in patients with pan-susceptible tuberculosis to 39% (nine of 23) in patients with pre-XDR or XDR tuberculosis. The adjusted odds ratio for mortality was 4·92 (95% CI 2·47–9·78) among undertreated patients, compared with appropriately treated patients. Interpretation: In seven countries with a high burden of tuberculosis, we observed discrepancies between drug resistance patterns obtained locally and WGS. The underdiagnosis of drug resistance resulted in inappropriate treatment and higher mortality. WGS can provide accurate and detailed drug resistance information required to improve the outcomes of drug-resistant tuberculosis in high-burden settings. Our results support WHO's call for point-of-care tests based on WGS. Funding: National Institutes of Allergy and Infectious Diseases, Swiss National Science Foundation, and Swiss National Center for Mycobacteria.

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.

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.

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.

Abstract Background Large sequence datasets are difficult to visualize and handle. Additionally, they often do not represent a random subset of the natural diversity, but the result of uncoordinated and convenience sampling. Consequently, they can suffer from redundancy and sampling biases. Results Here we present Treemmer, a simple tool to evaluate the redundancy of phylogenetic trees and reduce their complexity by eliminating leaves that contribute the least to the tree diversity. Conclusions Treemmer can reduce the size of datasets with different phylogenetic structures and levels of redundancy while maintaining a sub-sample that is representative of the original diversity. Additionally, it is possible to fine-tune the behavior of Treemmer including any kind of meta-information, making Treemmer particularly useful for empirical studies.

Abstract Background Bovine tuberculosis (BTB) is caused by Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. Mycobacterium bovis have been described to be responsible of most cases of bovine tuberculosis. Although M. tuberculosis, M. africanum and non-complex mycobacteria were isolated from cattle. In Morocco, so far, no molecular studies were conducted to characterize the strains responsible of BTB. The present study aims to characterize M. bovis in Morocco. The present study was conducted in slaughterhouses in Rabat and El Jadida. Samples were collected from 327 slaughtered animals with visible lesions suggesting BTB. Results A total of 225 isolates yielded cultures, 95% (n = 215) of them were acid-fast (AF). Sixty eight per cent of the AF positive samples were confirmed as tuberculous mycobacteria (n = 147), 99% of these (n = 146) having RD9 and among the latter, 98% (n = 143) positive while 2% (n = 3) negative for RD4 A total of 134 samples were analyzed by spoligotyping of which 14 were in cluster and with 41 different spoligotypes, ten of them were new patterns (23%). The most prevalent spoligotypes were SB0121, SB0265, and SB0120, and were already identified in many other countries, such as Algeria, Spain, Tunisia, the United States and Argentina. Conclusion The shared borders between Algeria and Morocco, in addition to the previous importation of cattle from Europe and the US could explain the similarities found in M. bovis spoligotypes. On the other hand, the desert of Morocco could be considered as an efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa. Our findings suggest a low level endemic transmission of BTB similar to other African countries. However, more research is needed for further knowledge about the transmission patterns of BTB in Morocco.

Transition bias, an overabundance of transitions relative to transversions, has been widely reported among studies of the rates and spectra of spontaneous mutations. However, demonstrating the role of transition bias in adaptive evolution remains challenging. In particular, it is unclear whether such biases direct the evolution of bacterial pathogens adapting to treatment. We addressed this challenge by analyzing adaptive antibiotic-resistance mutations in the major human pathogen Mycobacterium tuberculosis (MTB). We found strong evidence for transition bias in two independently curated data sets comprising 152 and 208 antibiotic-resistance mutations. This was true at the level of mutational paths (distinct adaptive DNA sequence changes) and events (individual instances of the adaptive DNA sequence changes) and across different genes and gene promoters conferring resistance to a diversity of antibiotics. It was also true for mutations that do not code for amino acid changes (in gene promoters and the 16S ribosomal RNA gene rrs) and for mutations that are synonymous to each other and are therefore likely to have similar fitness effects, suggesting that transition bias can be caused by a bias in mutation supply. These results point to a central role for transition bias in determining which mutations drive adaptive antibiotic resistance evolution in a key pathogen.

The Lineage 2–Beijing (L2–Beijing) sub-lineage of Mycobacterium tuberculosis has received much attention due to its high virulence, fast disease progression, and association with antibiotic resistance. Despite several reports of the recent emergence of L2–Beijing in Africa, no study has investigated the evolutionary history of this sub-lineage on the continent. In this study, we used whole genome sequences of 781 L2 clinical strains from 14 geographical regions globally distributed to investigate the origins and onward spread of this lineage in Africa. Our results reveal multiple introductions of L2–Beijing into Africa linked to independent bacterial populations from East- and Southeast Asia. Bayesian analyses further indicate that these introductions occurred during the past 300 years, with most of these events pre-dating the antibiotic era. Hence, the success of L2–Beijing in Africa is most likely due to its hypervirulence and high transmissibility rather than drug resistance.

INTRODUCTION:Recurrent tuberculosis (TB) is one of the main challenges in TB control. Genotyping based on Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) has been widely used to differentiate between relapse and reinfection, which are the two main causes of recurrent TB. There is a lack of data regarding the causes of TB recurrence in Georgia, and while differentiating between relapse and reinfection plays a key role in defining appropriate interventions, the required genotyping methodologies have not been implemented. The objective of this study was to implement MIRU-VNTR genotyping at the National Center for Tuberculosis and Lung Diseases (NCTBLD) and differentiate between relapse and reinfection in multidrug resistant (MDR-) TB patients from Tbilisi, Georgia. METHODS:Recurrent MDR tuberculosis cases from 2014-2016 diagnosed at NCTLD were included in the study when bacterial samples from both episodes were available. Genotyping based on the MIRU-VNTR 24 loci was implemented and used for differentiating between relapse and reinfection. Paired samples showing the same MIRU-VNTR pattern or one locus difference were classified as relapse, while two and more loci differences were treated as reinfection. Exact logistic regression was used to identify predictors of recurrence. RESULTS:Thirty two MDR-TB patients (64 samples) were included and MIRU-VNTR 24 typing was performed on the corresponding paired samples. Of the 32 patients, 25 (83.3%) were identified as relapse while 5 (16.7%) were due to re-infection. Patients with a history of incarceration were significantly associated with TB reinfection (p< 0.05). CONCLUSION:Recurrent TB in MDR patients in Georgia are mainly caused by relapse, raising concerns on the efficacy of the TB control program. An association between incarceration and reinfection likely reflects high levels of ongoing TB transmission in prisons, indicating the need for better TB infection control measures in these settings. Our results add to the rationale for implementing genotypic surveillance of TB more broadly to support TB control in Georgia.

BackgroundHuman 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 findingsHere 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.ConclusionsThis study provides novel insights into the MTBC lineages and the possible influence of pathogen-related factors on the TB epidemic in Tanzania.

Bacteria belonging to the genus Mycobacterium are predominantly responsible for pulmonary diseases; most notably Mycobacterium tuberculosis causes granulomatous pulmonary infections. Here we describe a novel slow growing mycobacterial species isolated from respiratory samples from five patients, four with underlying pulmonary disease. The isolates were characterized by biochemical and molecular techniques, including whole genome sequencing. Biochemical characteristics generally match those of M. marinum and M. ulcerans; however, the most striking difference of the new species is its ability to grow at 37°C. The new species was found to grow in human macrophages, but not amoebae, suggesting a pathogenic rather than an environmental lifestyle. Phylogenetic analysis reveals a deep-rooting relationship to M. marinum and M. ulcerans. A complete genome sequence was obtained through combining short and long-read sequencing, providing a genome of 5.6 Mb. The genome appears to be highly intact, syntenic with that of M. marinum, with very few insertion sequences. A vast array of virulence factors includes 283 PE/PPE surface-associated proteins, making up 10% of the coding capacity, and 22 non-ribosomal peptide synthase clusters. A comparison of six clinical isolates from the five patients shows that they differ by up to two single nucleotide polymorphisms, suggesting a common source of infection. Our findings are in accordance with the recognition of a new taxonomic entity. We propose the name M. basiliense, as all isolates were found in patients from the Basel area of Switzerland.

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.

Bacterial factors favoring the unprecedented multidrug-resistant tuberculosis (MDR-TB) epidemic in the former Soviet Union remain unclear. We utilized whole genome sequencing and Bayesian statistics to analyze the evolutionary history, temporal emergence of resistance and transmission networks of MDR Mycobacterium tuberculosis complex isolates from Karakalpakstan, Uzbekistan (2001–2006). One clade (termed Central Asian outbreak, CAO) dating back to 1974 (95% HPD 1969–1982) subsequently acquired resistance mediating mutations to eight anti-TB drugs. Introduction of standardized WHO-endorsed directly observed treatment, short-course in Karakalpakstan in 1998 likely selected for CAO-strains, comprising 75% of sampled MDR-TB isolates in 2005/2006. CAO-isolates were also identified in a published cohort from Russia (2008–2010). Similarly, the presence of mutations supposed to compensate bacterial fitness deficits was associated with transmission success and higher drug resistance rates. The genetic make-up of these MDR-strains threatens the success of both empirical and standardized MDR-TB therapies, including the newly WHO-endorsed short MDR-TB regimen in Uzbekistan.

Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67-5.47, p

BackgroundDifferent combinations of variable number of tandem repeat (VNTR) loci have been proposed for genotyping Mycobacterium tuberculosis complex (MTBC). Existing VNTR schemes show different discriminatory capacity among the six human MTBC lineages. Here, we evaluated the discriminatory power of a "customized MIRU12" loci format proposed previously by Comas et al. based on the standard 24 loci defined by Supply et al. for VNTR-typing of MTBC in Ghana.MethodOne hundred and fifty-eight MTBC isolates classified into Lineage 4 and Lineage 5 were used to compare a customized lineage-specific panel of 12 MIRU-VNTR loci ("customized MIRU-12") to the standard MIRU-15 genotyping scheme. The resolution power of each typing method was determined based on the Hunter-Gaston- Discriminatory Index (HGDI). A minimal set of customized MIRU-VNTR loci for typing Lineages 4 (Euro-American) and 5 (M. africanum West African 1) strains from Ghana was defined based on the cumulative HGDI.Results and conclusionAmong the 106 Lineage 4 strains, the customized MIRU-12 identified a total of 104 distinct genotypes consisting of 2 clusters of 2 isolates each (clustering rate 1.8%), and 102 unique strains while standard MIRU-15 yielded a total of 105 different genotypes, including 1 cluster of 2 isolates (clustering rate: 0.9%) and 104 singletons. Among, 52 Lineage 5 isolates, customized MIRU-12 genotyping defined 51 patterns with 1 cluster of 2 isolates (clustering rate: 0.9%) and 50 unique strains whereas MIRU-15 classified all 52 strains as unique. Cumulative HGDI values for customized MIRU-12 for Lineages 4 and 5 were 0.98 respectively whilst that of standard MIRU-15 was 0.99. A union of loci from the customised MIRU-12 and standard MIRU-15 revealed a set of customized eight highly discriminatory loci: 4052, 2163B, 40, 4165, 2165, 10,16 and 26 with a cumulative HGDI of 0.99 for genotyping Lineage 4 and 5 strains from Ghana.

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown.We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status.We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.

BackgroundMycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as "Beijing" has repeatedly been associated with drug resistance and has been emerging in some parts of the world. "Beijing" strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical "Beijing" spoligotyping pattern.Methods and findingsMTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this "Pseudo-Beijing" spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical "Beijing" strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC.ConclusionsWe provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of "Beijing" strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

Aims and objectives: Resistant-, multidrug resistant- (MDR) and even extensively drug resistant- (XDR) Mycobacterium tuberculosis complex (MTBC) strains have emerged worldwide and represent a serious challenge for global tuberculosis (TB) control. Interestingly, high rates of MDR TB have been associated with particular phylogenetic lineages of the MTBC such as the Beijing lineage and the strong clonal expansion of particular MDR strains in Eastern Europe. However, a systematic investigation of the evolution and cross border transmission of particular highly transmissible MDR strains in Eastern Europe has not been performed so far. Methods: To address this question, an in-depth analysis was performed of the population structure (based on 24 loci MIRU-VNTR-typing and spoligotyping) of strains from different settings in Eastern Europe, such as Uzbekistan, Turkmenistan, and Kazakhstan. Results: The results showed that two major clones of the Beijing lineage are the drivers of the MDR epidemic in Eastern Europe. In addition, retrospective investigation of an MDR strain collection from Germany using MIRU-VNTR and IS6110 RFLP patterns revealed that both clones were circulating for more than 15 years. In-depth analysis of genome characteristics confirmed the longitudinal transmission of the two major MDR clones and allowed the identification of particular beneficial combinations of mutations potentially compensating for the fitness defect of the MDR/XDR strains. Moreover, these allele combinations coincide with an enhanced transmissibility compared with other circulating MDR/XDR strain types and promote the selection of extremely drug resistant but also highly virulent MTBC strain variants. Conclusion: Taken together, these data argue strongly for an expansion of particular MDR Beijing clones in Eastern Europe that started at least 15 years ago. Evolution and selection over the past years has resulted in “hyper transmissible” MDR-TB strains that have the potential to spread rapidly and, thus, further complicate TB control.

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.