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The Haarlem family belongs to the Euro-American phylogenetic lineage of Mycobacterium tuberculosis and is one of the globally spread genotypes of this important human pathogen. In spite of the sporadic observations on drug resistance and peculiar virulence profile, Haarlem remains in the shade of other M. tuberculosis genotypes. I analyzed genotyping data of the Haarlem genotype in light of its pathogenic properties and relevant human migration, to gain insight into its origin, evolutionary history, and current spread. Central Europe is marked with a very high prevalence of both major Haarlem subclades ancestral H3/SIT50 and derived H1, jointly making 33-41% in Czechia, Austria, and Hungary. There is a declining gradient of Haarlem beyond central Europe with 10-18% in Italy, France, Belgium, 10-13% in the Balkan countries and Turkey. Placing the available genetic diversity and ancient DNA data within the historical context, I hypothesize that M. tuberculosis Haarlem genotype likely originated in Central Europe and its primary long-term circulation occurred within the area of the former Austria/Austria-Hungary Empire in the 14th-19th centuries. The genotype is not highly transmissible and its spread was driven by long-term human migration. The European colonial expansion (when accompanied by a sufficient volume of migration) was a vehicle of its secondary dissemination. I conclude that human migration and its lack thereof (but not strain pathobiology) was a major driving force that shaped the population structure of this global lineage of M. tuberculosis. At the same time, Haarlem strains appear over-represented in some ethnic groups which warrants in-depth experimental research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this study, we tested the skeletal human remains from the 18th - early 19th century Orthodox cemeteries in Irkutsk, Eastern Siberia, for tuberculosis-associated morphological alterations and Mycobacterium tuberculosis DNA. The morphologically studied bone collection included 591 individuals of mainly Caucasian origin. The molecular methods (IS6110-PCR and spoligotyping) suggested that at least four individuals (out of 15 TB-suspected, DNA-tested) were positive for the presence of M. tuberculosis DNA. All of them were males (3 maturus, 1 maturus senilis). Two of them date back to the second and third quarters of the 18th century, another to the last quarter of the 18th century, and the last one to the second half of the 19th century. The combined molecular analysis cautiously suggested presence of different strains and at least some of them represented not the currently predominant in Siberia Beijing genotype (M. tuberculosis East-Asian lineage) but strains of European origin. In conclusion, this study presented bioarchaeological and molecular evidence of tuberculosis in human skeletal remains from 18th-19th century Orthodox cemeteries in Irkutsk, Eastern Siberia. The samples are not M. bovis and represent human M. tuberculosis sensu stricto. Their precise phylogenetic identity is elusive but evokes the European/Russian origin of at least some isolates., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: . The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern., Results: . The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes)., Conclusions: . We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted., (© 2023. The Author(s).)

This study aimed to determine phenotypic and genotypic drug resistance patterns of Mycobacterium tuberculosis strains from children with tuberculosis (TB) in China and Russia, two high-burden countries for multi/extensively-drug resistant (MDR/XDR) TB. Whole-genome sequencing data of M. tuberculosis isolates from China ( n = 137) and Russia ( n = 60) were analyzed for phylogenetic markers and drug-resistance mutations, followed by comparison with phenotypic susceptibility data. The Beijing genotype was detected in 126 Chinese and 50 Russian isolates. The Euro-American lineage was detected in 10 Russian and 11 Chinese isolates. In the Russian collection, the Beijing genotype and Beijing B0/W148-cluster were dominated by MDR strains (68% and 94%, respectively). Ninety percent of B0/W148 strains were phenotypically pre-XDR. In the Chinese collection, neither of the Beijing sublineages was associated with MDR/pre-XDR status. MDR was mostly caused by low fitness cost mutations ( rpoB S450L, katG S315T, rpsL K43R). Chinese rifampicin-resistant strains demonstrated a higher diversity of resistance mutations than Russian isolates ( p = 0.003). The rifampicin and isoniazid resistance compensatory mutations were detected in some MDR strains, but they were not widespread. The molecular mechanisms of M. tuberculosis adaptation to anti-TB treatment are not unique to the pediatric strains, but they reflect the general situation with TB in Russia and China.

Introduction: The epidemiological situation of tuberculosis (TB) in Poland urges for its continuous and scrupulous monitoring. The objective of this study was to explore the genetic diversity of multidrug-resistant (MDR) and drug-susceptible (DS) Mycobacterium tuberculosis isolates from Poland with a combination of spoligotyping and high-resolution mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis. The results were placed in the Northern and Eastern Europe context., Methods: The study included 89 (39 MDR and 50 DS) M. tuberculosis isolates collected from as many patients between 2018 and 2021 in Poland. The analysis was done using spoligotyping, and MIRU-VNTR typing at 24 standard loci. The data were compared to those available on Poland and neighbors and global M. tuberculosis datasets., Results: The main identified families were Beijing (28.1%) and Haarlem (16.8%) while 34.8% of isolates were in the heterogeneous L4-unclassified group. Although the Beijing family was the most prevalent (61.5%) among MDR-TB cases, it accounted for only 2% of DS isolates. Among foreign-born patients, a higher ratio of MDR isolates were observed when compared with those who Poland-born (64.3% vs. 40%). Furthermore, all patients from the Former Soviet Union (FSU) countries were infected with MDR-TB., Discussion: Whereas DS M. tuberculosis population in Poland is dominated by L4 isolates, MDR isolates are mostly of the Beijing genotype. The rise in the prevalence of the Beijing isolates in Poland, coupled with high proportion of the Beijing genotype among foreign-born TB patients may reflect an ongoing transmission of this family, imported to Poland mainly from FSU countries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bakuła, Marczak, Bluszcz, Proboszcz, Kościuch, Krenke, Stakėnas, Mokrousov and Jagielski.)

The dramatic change in global health imposed by the Covid-19 pandemic has also impacted TB control. The TB incidence decreased dramatically not because of the improved situation but due to undertesting, reduced resources, and ultimately, substantially reduced detection rate. We hypothesized that multiple and partly counteracting factors could influence changes in the local Mycobacterium tuberculosis population. To test this hypothesis, we analyzed M. tuberculosis isolates collected in Western Siberia, Russia, before and during the Covid-19 pandemic. A total of 269 M. tuberculosis isolates from patients admitted at referral clinics were studied. The pre-pandemic and pandemic collections included 179 and 90 isolates, respectively. Based on genotyping, both pre-pandemic and pandemic samples are heavily dominated by the Beijing genotype isolates (95% and 88%) that were mostly MDR (80 and 68%). The high proportion of MDR isolates is due to the specific features of the studied collections biased towards patients with severe TB admitted at the National referral center in Novosibirsk. While no dramatic change was observed in the M. tuberculosis population structure in the survey area in Western Siberia during the Covid-19 pandemic in 2020-2021 compared to the pre-pandemic collection, still we note a certain decrease of the Beijing genotype and an increase in the proportion and diversity of the non-Beijing isolates. However, the transmissible and MDR Beijing B0/W148 did not increase its prevalence rate during the pandemic. More generally, the high prevalence rate of the Beijing genotype and its strong association with MDR both before and during the pandemic are alarming features of this region in Western Siberia, Russia., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Eastern Siberia (Russia) and Mongolia are borderline regions in Asia with a high incidence of tuberculosis (TB). In this study, we investigated the transborder transmission of Mycobacterium tuberculosis with a focus on endemic and epidemic clones and drug resistance. M. tuberculosis isolates (287 from Mongolia and 754 from Russia) were collected using cross-sectional population-based surveys between 2010 and 2016. The isolates were genotyped using 24 variable number of tandem repeat loci and by testing of the key markers to discriminate within the Beijing genotype. All isolates were divided into 427 mycobacterial interspersed repetitive units types that were assigned to Lineage 2 (Beijing) and Lineage 4 (Ural, Haarlem, Latin American-Mediterranean [LAM], S, unclassified). The Beijing genotype was dominant in both countries (69% in Russia, 75% in Mongolia). However, the Beijing isolates differed significantly between the countries, in terms of the identified subtypes. LAM was the most common non-Beijing genotype (11.1% in Mongolia and 14.9% in Russia) and LAM isolates mostly belonged to the LAM-RUS branch in both countries. The multidrug-resistance (MDR) rate was higher in Russia than in Mongolia among newly diagnosed patients: 29.4% versus 5.6% (p < .001). In Mongolia, the MDR rate was similar in Beijing (29.7%) and non-Beijing (27.5%) genotypes. In Russia, a higher MDR rate was observed in (i) Beijing compared with non-Beijing (48.7% versus 38.3%, p = .03) and (ii) Beijing B0/W148 compared with Beijing Central Asian/Russian (63.4% versus 37.3%, p < .001). In conclusion, the M. tuberculosis population structure in Mongolia was shaped by mainly historical interaction with China (dominance of the Beijing genotype) and Northern Eurasia (presence of the LAM-RUS branch). In contrast, the transborder transmission of M. tuberculosis since the 1990s between Mongolia and its neighbours has been negligible, and the adverse trends of MDR-TB in Russia did not impact the current situation in Mongolia., (© 2022 Wiley-VCH GmbH.)

Nigeria ranks 1st in Africa and 6th globally with the highest burden of tuberculosis (TB). However, only a relatively few studies have addressed the molecular epidemiology of Mycobacterium tuberculosis in this country. The aim of this work was to analyze the genetic structure of drug-resistant (DR) M. tuberculosis population in the Plateau State (central Nigeria), with the results placed in the broader context of West Africa. The study sample included 67 DR M. tuberculosis isolates, recovered from as many TB patients between November 2015 and January 2016, in the Plateau State. The isolates were subjected to spoligotyping and MIRU-VNTR typing. A total of 20 distinct spoligotypes were obtained, split into 3 clusters (n = 50, 74.6%, 2-33 isolates per cluster) and 17 (25.4%) unique patterns. The Cameroon clade was the largest lineage (62.7%) followed by T (28.3%), LAM (3%), and Haarlem (3%) clades. Upon MIRU-VNTR typing, the isolates produced 31 profiles, i.e. 7 clusters (n = 43, 64.2%, 2-17 isolates per cluster) and 24 singletons. A combined spoligotyping and MIRU-VNTR typing analysis showed 20.9% of the cases clustered and estimated the recent transmission rate at 11.9%. In conclusion, two lineages, namely Cameroon, and T accounted for the majority (91%) of cases. No association was observed between the most prevalent Cameroon lineage and drug resistance, including multidrug resistant (MDR) phenotype, or any of the patient demographic characteristics., Competing Interests: The authors have declared that no competing interests exist.

Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidences of tuberculosis in the country. Retrospective mycobacterial interspersed repetitive unit/variable number of tandem repeats analysis of 42 isolates collected between January and August 2018 allowed to identify a cluster (Beijing A) with 17 (40.5%) Beijing isolates. Subsequent prospective strain-specific PCR-based surveillance from September 2019 to March 2020 confirmed the predominance of the Beijing A strain (44.1%) in this province. Whole-genome sequencing revealed higher-than-expected diversity within the cluster, suggesting long-term prevalence of this strain and low number of cases caused by recent transmission. The Beijing A strain belongs to the Asian African 3 (Bmyc13, L2.2.5) branch of the modern Beijing sublineage, with their closest isolates corresponding to cases from Vietnam, probably introduced in Panama between 2000 and 2012., (© 2020 Wiley-VCH GmbH.)

Euro-American lineage (Lineage 4) of Mycobacterium tuberculosis comprises genetically and geographically diverse families that differ in their clinical and/or epidemiological capacities. Due to the characteristic structure of the CRISPR locus (presence of almost all 43 classical spacers except for deleted signals 33-36), spoligotype SIT53 takes the basal position in the evolution of this lineage. In the SITVIT database, it is assigned to the "ill-defined" T family and T1 subfamily. Here, I analyzed the phylogenetic diversity of SIT53 isolates and discussed interconnected terminological issues concerning M. tuberculosis population structure. The 24-MIRU-VNTR profiles of 266 SIT53 isolates from Europe, Asia, Africa, and South America were submitted to the phylogenetic analysis jointly with reference profiles of different families from MIRU-VNTRplus database. Under this analysis, SIT53 isolates were clustered within different and distant families such as Ghana, NEW-1 (L4.5), TUR (L4.2.2.1), etc whereas many remained unclassified within L4. This confirms the evolutionarily basal position of this spoligotype and in its turn, this demonstrates that SIT53 does not correspond to any particular family of M. tuberculosis. Instead, different SIT53 subpopulations with evolutionarily stable and unchanged CRISPR locus gave rise to different and distant families that in many instances evolved through long-term allopatric evolution., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis)., Results: Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years., Conclusions: The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.

The aim of this study was to perform a molecular characterization of Mycobacterium tuberculosis strains circulating in one of the "closed" Russian cities under conditions of the limited population migration and high HIV coinfection rate. We analyzed 109 M. tuberculosis isolates recovered from TB patients in the Novouralsk municipality in the Ural area of Russia; 38.5% were from HIV coinfected TB patients and 19.3% patients were former prison inmates. The Beijing genotype was predominant (78.9%) while 57.8% and 17.4% of isolates belonged to the Beijing B0/W148 and Beijing 94-32-clusters, respectively. An atypical allele of the QUB26 VNTR locus (2 repeat units) was detected in 11 of 63 Beijing B0/W148 isolates. The non-Beijing isolates were subdivided into nine spoligotypes of the four genetic families (Ural, LAM, Haarlem, T), SIT35/Ural being the largest group (n = 9; 8.3%). Multidrug resistance (MDR) was detected in 63.6% and 83.7% of isolates from newly diagnosed and previously treated patients, respectively. Almost all isolates of the B0/W148-cluster were MDR (92.1%) compared to the Beijing 94-32-cluster (47.4%). No association was found between HIV status of patients and MDR-TB or particular genetic cluster. A combined contact and molecular investigation confirmed three family foci; in two of them, Ural SIT35 and Beijing B0/W148 strains were transmitted from HIV-infected sons to their fathers. To conclude, M. tuberculosis population in Novouralsk features an exceptionally high prevalence of the strongly MDR Beijing B0/W148-cluster and emergence of the B0/W148 substrain with unusual QUB26 allele. This situation was likely synergistically shaped by the limited population migration, high prevalence of the HIV coinfection and high proportion of the former prisoners. The existing organizational approaches to prevent TB transmission are insufficient and require a serious revision., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Albania is a Balkan country with moderate to low incidence of tuberculosis (TB) and very low prevalence of drug resistant TB. Here, we analyzed a country-wide multi-year Mycobacterium tuberculosis collection in order to detect possible dynamic trends of TB in Albania, with a focus on drug resistance and endemic/epidemic clones. In total, 743 isolates collected in 2007 to 2011 were divided into 107 spoligotypes and 351 MIRU-types. Based on the MIRU-VNTR phylogenetic analysis, the isolates were assigned to the following lineages/families: animal ecotypes (5 M. bovis and 2 M. caprae isolates), Lineage 2 (5 Beijing isolates), Lineage 3 (1 CAS-Delhi isolate) and, mostly and overwhelmingly, Lineage 4 (Cameroon, Uganda, Ghana and related; NEW-1-related; Ural, Haarlem, LAM, S, TUR; and unclassified isolates). Most of the isolates (452/743) were intermediately located on the global VNTR tree and did not cluster with any reference profile; they were distantly related to different families within Lineage 4 and we designated them as "unclassified L4" isolates. The significantly higher proportion of drug resistance was observed in (i) Beijing genotype compared to all other isolates (60%, P = .008), (ii) "unclassified L4" compared to all other isolates (13.9%, P = .04) and (iii) SIT2936 compared to other "unclassified L4" (34.3%, P = .0006). Analysis of the yearly collections revealed (i) some decrease of the large heterogeneous "unclassified L4" from 65% to 57%; (ii) steadily increasing gradient of LAM from 3.4 to 13.3%; (iii) stable prevalence of Haarlem (15-20%); and (iv) decrease of TUR with only 1.1% in 2011. Most of the LAM (33/49) and Beijing (3/5) isolates belonged to the VNTR types specific for Russia and former Soviet Union countries. To conclude, our results highlight a peculiar nature of M. tuberculosis population in Albania that is dominated by local and unclassified genotypes within Lineage 4, and also features European genotypes and epidemically relevant clones originating from the former Soviet Union countries. At the same time, these imported clones remain drug susceptible and prevalence of drug resistance on a whole is low., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The 2nd St. Petersburg Symposium on Tuberculosis and Mycobacteria: Molecular Approach, was held in St. Petersburg, Russia on 5-6 December 2018. A special issue of Infection, Genetics and Evolution will publish articles based on the selected presentations. In this paper, I will discuss some of the hot topics of molecular mycobacteriology highlighted at this meeting that I had the pleasure to organize and honor to chair. The symposium addressed interrelated fundamental and applied issues of modern mycobacteriology such as molecular evolution and phylogenomics, host-microbe interactions and pathogenesis, coevolution of M. tuberculosis with humans, new genomic and postgenomic technologies. Molecular methods for TB diagnostics and drug resistance detection are supported by WHO and whole genome/next generation sequencing presents a comprehensive approach. At the same time, cost and implementation of new methods for direct analysis of clinical samples and/or in low-resource settings remain a great challenge. A due attention was also given to the medically important nontuberculous mycobacteria. Assessment of spectrum of the circulating mycobacterial species in the Russian Federation and the countries of the European Union was presented and the underlying reasons of the observed diversity were discussed. To conclude, the symposium became a multidisciplinary event that was useful to promote networking and exchange of knowledge and experience. The next (third) symposium was planned to be organized in 2021., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Ural genetic family is a part of the Euro-American lineage of Mycobacterium tuberculosis and is endemic in Northern Eurasia (former Soviet Union [FSU]). These strains were long described as drug susceptible and of low virulence, but recent studies reported an increasing circulation of the multidrug-resistant (MDR) and extensively drug-resistant (XDR) Ural strains. Here, we analyzed all publicly available whole genome sequence data of Ural genotype isolates, in order to elucidate their phylogenomic diversity with a special focus on MDR and potentially epidemic clones., Results: A total of 149 M. tuberculosis genomes of Ural isolates from FSU countries were mined from the GMTV database and TB-ARC project. We identified 6002 variable amino acid positions that were assessed for functional significance and used to build ML, NJ trees and for Bayesian TMRCA estimation. Three robust monophyletic clades were identified: Clade A (31 isolates from Russia, Belarus, Moldova), Clade B (52 isolates from Russia), and Clade C (37 isolates from Moldova, 2 from Belarus). Clade C was significantly associated with XDR or pre-XDR status compared to the pooled Clades A and B (33/39 versus 5/83, P < 0.0001). Time of origin was estimated for Clade A at 77.7-137 years ago and for Clade B at 56.3-99.2 years ago compared to the significantly more recent origin for Clade C. in silico spoligotyping identified signatures specific of the Clade A (spoligotype SIT35), and Clades B and C (both SIT262)., Conclusions: A genetically compact and evolutionarily young Ural Clade C, likely originated after collapse of the Soviet Union, and reached epidemic proportions in Moldova in the last 20 years. This epidemic pre-XDR clone (mostly rifampin, isoniazid and kanamycin resistant) is characterized by a specific combination of mutations: KatG Ser315Thr, fabG1 -15C > T, RpoB Ser450Leu, RpsL Lys88Arg, eis -12G > A and EmbB Ser297Ala/T > G. Its further dissemination may occur towards both Russia and European Union and should be taken into consideration by health authorities. The identified spoligotyping signatures can serve for rapid preliminary detection and surveillance of the more hazardous pre-XDR associated strains of the Ural family, both in populations from countries of their endemic circulation and migrant communities.

CRISPR-spoligotyping and MIRU-VNTR typing, SITVIT&#95;WEB and MIRU-VNTRplus are the methods and online resources most widely used for Mycobacterium tuberculosis genotype family assignment and clustering analysis. They have been proven invaluable for molecular epidemiological studies of this important human pathogen in setting up the terminology and classification framework. However, they are inherently limited by insufficient knowledge of evolution of the targeted genome loci (especially, CRISPR). The situation is aggravated by the dogmatic, iconographic perception of these increasingly user-friendly online tools. Here, I present a critical essay on hot practical aspects related to the use of SITVIT&#95;WEB and MIRU-VNTRplus, in particular, partly inadequate (sub)clade assignment due to imperfect decision rules, partly outdated methodological options offered to the users that permit to build scientifically unsound phylogenies from spoligotyping data. A confusing terminology, misclassification and false clustering are not abstract issues but make a scientific discussion meaningless, and I propose some courses for improvement., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Whole-genome analysis of Mycobacterium tuberculosis isolates collected in Russia (N = 71) from patients with tuberculous spondylitis supports a detailed characterization of pathogen strain distributions and drug resistance phenotype, plus distinguished occurrence and association of known resistance mutations. We identify known and novel genome determinants related to bacterial virulence, pathogenicity, and drug resistance.

Due to its rapid spread and association with the numerous outbreaks, the global spread of East Asian lineage of Mycobacterium tuberculosis strains presents a global concern. Although there were many attempts to describe its population structure, no consensus has been reached yet. To define unbiased classification that will facilitate future studies of this lineage, we analyzed the performance and congruence of eight different genotyping schemes based on phylogenetic analysis of 1,398 strains from 32 countries using whole-genome sequencing (WGS) data. We confirm that East Asian lineage comprises two major clades, designated proto-Beijing, which harbors unusual 43-signal spoligoprofile, and Beijing, with well-known spoligoprofile (deleted signals from 1 to 34). We show that different genotyping methods give high consistency results in description of ancient Beijing strains while the classification of modern Beijing strains is significantly divergent due to star-shaped phylogeny. Using WGS data we intersect different studies and for the first time provide balanced classification with well-defined major groups and their genetic markers. Our reconstructed phylogenetic tree can also be used for further analysis of epidemiologically important clusters and their ancestors as well as white spots of unclassified strains, which are prospective areas of research.

Here, I review the population structure and phylogeography of the two contrasting families of Mycobacterium tuberculosis, Beijing and Ural, in the context of strain pathobiology and human history and migration. Proprietary database (12-loci MIRU-VNTR profiles of 3067 Beijing genotype isolates) was subjected to phylogenetic and statistical analysis. The highest rate (90%) and diversity (HGI 0.80-0.95) of the Beijing genotype in North China suggest it to be its area of origin. Under VNTR-based MDS analysis the interpopulation genetic distances correlated with geography over uninterrupted landmasses. In contrast, large water distances together with long time generated remarkable outliers. Weak and less expected affinities of the distant M. tuberculosis populations may reflect hidden epidemiological links due to unknown migration. Association with drug-resistance or increased virulence/transmissibility along with particular human migration flows shape global dissemination of some Beijing clones. The paucity of data on the Ural genotype prevents from high-resolution analysis that was mainly based on the available spoligotyping data. The North/East Pontic area marked with the highest prevalence of the Ural family may have been the area of its origin and primary dispersal in Eurasia. Ural strains are not marked by increased pathogenic capacities, increased transmissibility and association with drug resistance (but most recent reports describe an alarming increase of MDR Ural strains in some parts of eastern Europe and northwestern Russia). Large-scale SNP or WGS population-based studies targeting strains from indigenous populations and, eventually, analysis of ancient DNA will better test these hypotheses. Host genetics factors likely play the most prominent role in differential dissemination of particular M. tuberculosis genotypes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility., Objective: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17)., Methods: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells., Results: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group., Conclusions: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Latin-American-Mediterranean (LAM) family is among the largest, diverse and intriguing within Mycobacterium tuberculosis. Widespread in some world regions, as justly reflected by its name, LAM remains in the shadow of the Beijing and East-African Indian families in East Asia. Here, we applied evolutionarily robust markers to the spoligotyped collection of 699 M. tuberculosis clinical isolates recovered from tuberculosis (TB) patients in different provinces across China between 2005 and 2013. As a result, only two isolates were assigned to LAM, hence we conclude that this family has a negligible impact on TB transmission in China. Interestingly, these two isolates represented two grand sublineages within LAM: RD115 and RD-Rio/RD174. Our results highlight the great caution to be taken when interpreting spoligotyping profiles with large deleted blocks. Here, three geographically distant Chinese isolates of spoligotype SIT803 were excluded from LAM. This is in clear contrast with described SIT803 isolates of the LAM family in Russia and Georgia (Mokrousov et al., 2014; Niemann et al., 2010). Interestingly, a drug resistant isolate of the Ibero-American LAM RD-Rio sublineage was recovered from TB patient of Tibetan ethnicity, a resident in the Lhasa district in Tibet. An additional in-depth analysis by whole genome sequencing might help to understand the evolutionary history of this isolate and its possible route to the rural setting of the Tibetan highlands., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.

Background: Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome., Description: Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes., Conclusions: Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.

Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a "successful" clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.

An interaction of different human alleles and endemic bacterial strains may be clinically manifested as different outcome of the disease in different hosts infected with the same genotype. The primary objective of this study was to investigate this issue in the model of Mycobacterium tuberculosis and human DC-SIGN encoding CD209 promoter SNP (rs4804803) in Russian Siberian population. We sought to find a possible combination of M. tuberculosis lineage and human host allele/genotype correlating with unfavorable outcome of the disease. The 101 paired DNA samples from patients with pulmonary TB (human and M. tuberculosis DNA) were studied by 12-loci MIRU-VNTR typing (M. tuberculosis strains) and CD209 -336 A/G typing (human DNA). Ninety autopsy DNA samples as a source of human and mycobacterial nucleic acids from persons who died from TB were also subjected to the same genotyping procedures. A human control group consisted of 177 healthy individuals. The Beijing genotype was more frequently identified in autopsy versus patient samples, in 70.0% and 51.5%, respectively (χ(2)=6.06, P=0.01). Regarding other M. tuberculosis genetic families, no significant difference in LAM family distribution among patient strains and autopsy samples has been found. In contrast, Ural genotype was significantly less frequently detected in the autopsy samples (χ(2)=6.12, P=0.01). Allelic and genotypic frequencies of the CD209 -336A/G did not differ significantly under global comparison when contrasting controls versus patients versus autopsy samples. However intriguing and contrasting significant associations were found in the male subgroup under M. tuberculosis genotype-stratified comparisons. Firstly, male carriers of -336AA genotype were more frequently infected with Beijing genotype (χ(2)=5.2, P=0.02). Secondly and remarkably, this association was inverted in the autopsy sample: male carriers of -336AA genotype died less frequently due to TB caused by a Beijing rather than a non-Beijing strain (χ(2)=5.37, P=0.02). In conclusion, we hypothesize that although carriers of CD209 -336A allele are more sensitive to infection with a Beijing strain, a combination of human CD209 -336G allele and M. tuberculosis Beijing genotype leads more frequently to the lethal outcome in pulmonary TB male patients in Russian (Caucasian) population., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The application of genotyping tools to the analysis of tuberculosis (TB) has allowed us to identify clinical isolates of Mycobacterium tuberculosis to strain level. M. tuberculosis fingerprinting has been applied at different levels: a) in the laboratory, to optimize identification of cross-contamination events which can lead to a false diagnosis; b) in the patient, to determine whether recurrences are due to reactivations or exogenous reinfections or to identify cases coinfected by more than one strain; c) at the micropopulation level, to identify clusters of cases infected by the same strains (recent transmission) and to differentiate them from orphan cases that are most probably due to reactivations; and d) at the macropopulation level, to define the global distribution of M. tuberculosis lineages, to monitor the international spread of high-risk strains, and to explore the evolutionary features of M. tuberculosis. In recent years, important methodological and strategic advances have been applied at these different levels of analysis. Rather than provide an exhaustive review, the present study focuses on specific advances in micropopulation and macropopulation analysis., (Copyright © 2011 Elsevier España S.L. All rights reserved.)

The local specificity of bacterial clones may be explained by long-term presence or recent importation/fast dissemination in an area. Mycobacterium tuberculosis spoligotype ST125, noticeably prevalent among Bulgaria-specific spoligotypes, has a characteristically 'abridged' profile and an uncertain clade position [Latin-American-Mediterranean (LAM)/S]. A comparison with the SITVIT2 database (Institut Pasteur de Guadeloupe) demonstrated its high gradient in Bulgaria (14.3%) compared with the negligible presence in the rest of the world. Further typing of all available Bulgarian ST125 strains revealed that they: (i) monophyletically clustered in 21-mycobacterial interspersed repetitive units (MIRU)-loci tree of all Bulgarian strains; (ii) grouped closely with the ST34 spoligotype, a prototype of the S family; and (iii) did not harbor a LAM-specific IS6110 insertion. Comparison of the 21-MIRU-based network with geographic data revealed a complex dissemination pattern of ST125 in Bulgaria. Interestingly, this variable number of tandem repeats (VNTR) network remarkably corroborated with a recent hypothesis of single repeat loss as the primary mode of evolution of VNTR loci in M. tuberculosis. In conclusion, M. tuberculosis spoligotype ST125 is phylogeographically specific for Bulgaria. This spoligotype was not associated with drug resistance or increased transmissibility; its prevalence in Bulgaria can rather be attributed to the historical circulation in the country, having led, speculatively, to adaptation to the local human population.

The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100,000 in 2000 to 134/100,000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG. A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.

Several studies have suggested that genetic factors may affect the susceptibility of a population to tuberculosis, and it has been found that P2X7 is linked to an increased risk for tuberculosis in some West African, Southeast Asian, North American, and North European populations. To explore the potential role of P2X7 in the susceptibility to tuberculosis among members of the Chinese Han population, we evaluated the association of the 1513A-->C and -762T-->C polymorphisms in P2X7 with the risk for tuberculosis. PCR amplification of genomic DNA was followed by restriction fragment length polymorphism analysis, and allele-specific PCR was used. We found no significant differences in the genotypic and allelic frequencies of 1513A-->C polymorphisms in 96 patients with tuberculosis compared with 384 control subjects [P=0.856 and 0.316, respectively; odds ratio (OR) for the C allele=0.976; 95% confidence interval (CI)=0.755-1.262]. Similarly, no significant association was found between the -762T-->C polymorphism and tuberculosis (P=0.102 and 0.095 for the patients and controls, respectively; OR for the C allele=0.924; 95% CI=0.847-1.010). Thus, our analysis of P2X7 showed that the 1513A-->C and -762T-->C polymorphisms did not appear to be associated with the susceptibility of the Chinese Han population to tuberculosis.

The Beijing genotype of Mycobacterium tuberculosis has been shown in many settings to be hypervirulent and associated with multi-drug resistance. Its presently global and rapid dissemination makes it an important issue of public health. Here, I present a significantly enlarged update of the MIRU-VNTR global database of the M. tuberculosis Beijing genotype (11 loci). I further attempted to link the observed mycobacterial diversity with relevant events of the known human history. Large water masses have been the most efficient and drastic generators of the genetic divergence between human populations. The same situation appears true also for M. tuberculosis, which general diversity pattern amazingly resembles that of its human host. At the same time, less expected affinities observed between distant populations of M. tuberculosis may reflect hidden patterns of human migrations or yet unknown epidemiological links between distant regions.

The Beijing genotype is a globally spread lineage of Mycobacterium tuberculosis. In Russia, these strains constitute half of the local population of M. tuberculosis; they are associated with multidrug resistance and show increased transmissibility. Here, we analyzed traditional and new markers for the rapid and simple genotyping of the Beijing strains. A representative sample of 120 Beijing genotype strains was selected from a local IS6110-restriction fragment length (RFLP) database at the St. Petersburg Pasteur Institute. These strains were subjected to variable-number tandem-repeat (VNTR) typing using 24 loci of a newly proposed format and three hypervariable (HV) loci (QUB-3232, VNTR-3820, and VNTR-4120). Ten of the 27 VNTR loci were monomorphic, while five loci, MIRU26, QUB-26, QUB-3232, VNTR-3820, and VNTR-4120, were the most polymorphic (Hunter Gaston index, >0.5). VNTR typing allowed us to differentiate between two large IS6110-RFLP clusters known to be prevalent across the entire country (clusters B0/W148 and A0) and identified in 27 and 23% of strains, respectively, in the Beijing genotype database. The B0/W148 strains were grouped closely in the VNTR dendrogram and could be distinguished by a characteristic signature of the loci MIRU26 and QUB-26. Consequently, this clinically important IS6110-RFLP variant, B0/W148, likely presents a successful clonal group within the M. tuberculosis Beijing lineage that is widespread in Russia. To conclude, the IS6110-RFLP method and VNTR typing using a reduced set of the most polymorphic loci complement each other for the high-resolution epidemiological typing of the M. tuberculosis Beijing genotype strains circulating in or imported from Russia.

We report results of the first study on the molecular basis of drug resistance in Mycobacterium tuberculosis strains currently circulating in Bulgaria. The study panel consisted of 133 (including 37 drug-resistant) isolates recovered from newly diagnosed, adult pulmonary TB patients from different regions of Bulgaria in 2005--2006. Three types of the rpoB mutations were found in 20 of 27 RIF-resistant isolates; rpoB S531L was the most frequent. Eleven (48%) of 23 INH-resistant isolates had katG S315T mutation. inhA -15C > T mutation was detected in one INH-resistant isolate (that also had katG315 mutation) and three INH-susceptible isolates. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. Comparison with spoligotyping and 24-VNTR locus typing data suggested that emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any specific spoligotype or MIRU-VNTR genotype.

The development of tuberculosis (TB) infection in humans depends on the mycobacterial strain and the human host, and is multigenically controlled in both. ATP ligation of P2X(7) receptors expressed on human macrophages infected with mycobacteria induces cell death and subsequent loss of intracellular bacterial viability. This study analysed the allelic distribution of two single-nucleotide polymorphisms (SNPs) in the P2RX7 gene in the Slavic population of the St Petersburg area of Russia. Analysis of the -762 C/T P2RX7 promoter SNP revealed no significant association between pulmonary TB patients and control subjects (3x2 chi(2)=3.2, 1 d.f., P=0.2). The -762C allele was highly and almost equally represented in both groups in this study (68.2 % in patients and 69.3 % in controls). This result differs strikingly from a Gambian study where this allele was found in only 7 and 12 % of pulmonary TB patients and controls, respectively [Li, C. M., Campbell, S. J., Kumararatne, D. S., Bellamy, R., Ruwende, C., McAdam, K. P. W. J., Hill, A. V. S. & Lammas, D. A. (2002). J Infect Dis 186, 1458-1462]. In contrast, the frequency of the C allele at position 1513 in exon 13, resulting in a loss of P2X(7) function, was significantly higher among pulmonary TB patients in this study (P=0.02). Thus, analysis of the P2X(7) receptor gene in the Russian Slavic population showed that the 1513C allele, acting dominantly, is a possible risk factor for clinical TB, whereas the -762 P2RX7 polymorphism did not appear to be associated with human susceptibility to TB.

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7&#95;TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.

The Mycobacterium tuberculosis Beijing genotype strains appear to be hypervirulent and associated with multidrug-resistant tuberculosis. Therefore, the development of a both rapid and simple method to detect the M. tuberculosis Beijing genotype is of clinical interest per se. Previously, we described a simple and fast approach to detect the Beijing genotype based on IS6110 inverse-PCR typing. Here, we evaluated this method against a large, diverse, and recent collection of strains. The study sample included 866 M. tuberculosis strains representing but not limited to the regions in Russia, Europe, and East Asia where the Beijing genotype is endemic. Based on a spoligotyping method, 408 strains were identified as Beijing genotypes; they were additionally subdivided into ancient and modern sublineages based on the analysis of the NTF locus. All strains were further subjected to the IS6110-based inverse PCR. All of the Beijing genotype strains were found to have identical two-band (ancient sublineage) or three-band (modern sublineage) profiles that were easily recognizable and distinct from the profiles of the non-Beijing strains. Therefore, we suggest using IS6110-based inverse-PCR typing for the correct identification of the Beijing genotype and its major sublineages. The method is fast and inexpensive and does not require additional experiments but instead is implemented in the routine typing method of M. tuberculosis.

Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database., Results: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network., Conclusion: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11708 patterns from as many clinical isolates originating from more than 90 countries. The 11708 spoligotypes were clustered into 813 shared types. A total of 1300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Russia is a high-burden area for multidrug-resistant tuberculosis (MDR-TB). Here, we studied the epidemiological situation and drug resistance patterns of Mycobacterium tuberculosis in the Omsk region in Western Siberia. M. tuberculosis isolates (n = 851) were recovered from newly diagnosed TB patients in 2021. The isolates were tested by bacteriological and molecular methods, and long-term epidemiological data were analyzed. The TB incidence dec, this is not variablereased from 93.9 in 2012 to 48.1 in 2021, per 100,000 population, but the primary MDR-TB rate increased from 19.2% to 26.4%. The destructive forms of tuberculosis accounted for 37.8% of all cases, while 35.5% of patients were smear-positive. Of all isolates tested, 55.2% were culture-positive, of which 94.5% were further tested for phenotypic drug resistance and associated mutations. More than half (53.4%) of isolates were drug-resistant, 13.9% were monoresistant and 67.9% were MDR. Among MDR isolates, 40.4% were pre-XDR, and 19.2% were XDR. The spectrum of drug resistance included second-line drugs (new-generation fluoroquinolones, linezolid), which significantly increase the risk of an adverse outcome in patients. In conclusion, our results highlight the critical importance of monitoring drug resistance in circulating M. tuberculosis strains emerging due to ineffective treatment and active transmission. [ABSTRACT FROM AUTHOR]

The Beijing genotype is the main family of Mycobacterium tuberculosis in Russia. We analyzed its diversity and drug resistance in provinces across Northwestern Russia to identify the epidemiologically relevant Beijing strains. The study collection included 497 isolates from newly-diagnosed tuberculosis (TB) patients. Bacterial isolates were subjected to drug-susceptibility testing and genotyping. The Beijing genotype was detected in 57.5% (286/497); 50% of the Beijing strains were multidrug-resistant (MDR). Central Asian/Russian and B0/W148 groups included 176 and 77 isolates, respectively. MDR was more frequent among B0/W148 strains compared to Central Asian/Russian strains (85.7% vs. 40.3%, p < 0.0001). Typing of 24 minisatellite loci of Beijing strains revealed 82 profiles; 230 isolates were in 23 clusters. The largest Central Asian/Russian types were 94-32 (n = 75), 1065-32 (n = 17), and 95-32 (n = 12). B0/W148 types were 100-32 (n = 59) and 4737-32 (n = 5). MDR was more frequent in types 1065-32 (88.2%), 100-32 (83.1%), and 4737-32 (100%). In contrast, type 9391-32 (n = 9) included only drug-susceptible strains. To conclude, M. tuberculosis Beijing genotype is dominant in Northwestern Russia, and an active transmission of overwhelmingly MDR B0/W148 types explains the reported increase of MDR-TB. The presence of MDR-associated minor variants (type 1071-32/ancient Beijing and Central Asia Outbreak strain) in some of the studied provinces also requires attention. [ABSTRACT FROM AUTHOR]

We performed synthesis of new nitrofuranyl amides and investigated their anti-TB activity and primary genetic response of mycobacteria through whole-genome sequencing (WGS) of spontaneous resistant mutants. The in vitro activity was assessed on reference strain Mycobacterium tuberculosis H37Rv. The most active compound 11 was used for in vitro selection of spontaneous resistant mutants. The same mutations in six genes were detected in bacterial cultures grown under increased concentrations of 11 (2×, 4×, 8× MIC). The mutant positions were presented as mixed wild type and mutant alleles while increasing the concentration of the compound led to the semi-proportional and significant increase in mutant alleles. The identified genes belong to different categories and pathways. Some of them were previously reported as mediating drug resistance or drug tolerance, and counteracting oxidative and nitrosative stress, in particular: Rv0224c, fbiC, iniA, and Rv1592c. Gene-set interaction analysis revealed a certain weak interaction for gene pairs Rv1592–Rv1639c and Rv1592–Rv0224c. To conclude, this study experimentally demonstrated a multifaceted primary genetic response of M. tuberculosis to the action of nitrofurans. All three 11-treated subcultures independently presented the same six SNPs, which suggests their non-random occurrence and likely causative relationship between compound action and possible resistance mechanism. [ABSTRACT FROM AUTHOR]

The synthesis of 20 arylidenecamphors and 15 pyrimidines with camphane skeleton is described in the current report. A modified method for preparation of sterically hindered 2‐aminopyrimidines in two steps was demonstrated. The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed different MIC values (up to 0.91 μM for ketone 39). Compound 35 demonstrated moderate (8.23 μM), but sustainable activity toward a collection of drug‐resistant M. tuberculosis strains. Many of the compounds (especially among 2‐aminopyridines 42–56) exhibited good to excellent activity against different strains of pathogenic bacteria and fungi (MIC up to 0.60 μM for compound 50), compared with reference antibiotics. Many of the newly designed compounds possess also in vitro cytotoxicity. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis population in Russia is dominated by the notorious Beijing genotype whose major variants are characterized by contrasting resistance and virulence properties. Here we studied how these strain features could impact the progression of pulmonary tuberculosis (TB) concerning clinical manifestation and lethal outcome. Results: The study sample included 548 M. tuberculosis isolates from 548 patients with newly diagnosed pulmonary TB in Omsk, West Siberia, Russia. Strains were subjected to drug susceptibility testing and genotyping to detect lineages, sublineages, and subtypes (within Beijing genotype). The Beijing genotype was detected in 370 (67.5%) of the studied strains. The strongest association with multidrug resistance (MDR) was found for epidemic cluster Beijing B0/W148 (modern sublineage) and two recently discovered MDR clusters 1071–32 and 14717–15 of the ancient Beijing sublineage. The group of patients infected with hypervirulent and highly lethal (in a mouse model) Beijing 14717–15 showed the highest rate of lethal outcome (58.3%) compared to Beijing B0/W148 (31.4%; P = 0.06), Beijing Central Asian/Russian (29.7%, P = 0.037), and non-Beijing (15.2%, P = 0.001). The 14717–15 cluster mostly included isolates from patients with infiltrative but not with fibrous-cavernous and disseminated TB. In contrast, a group infected with low virulent 1071–32-cluster had the highest rate of fibrous-cavernous TB, possibly reflecting the capacity of these strains for prolonged survival and chronicity of the TB process. Conclusions: The group of patients infected with hypervirulent and highly lethal in murine model 14717–15 cluster had the highest proportion of the lethal outcome (58.3%) compared to the groups infected with Beijing B0/W148 (31.4%) and non-Beijing (15.2%) isolates. This study carried out in the TB high-burden area highlights that not only drug resistance but also strain virulence should be considered in the implementation of personalized TB treatment. [ABSTRACT FROM AUTHOR]

Ancient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster. [ABSTRACT FROM AUTHOR]