Origin and dispersal of the Mycobacterium tuberculosis Haarlem genotype: Clues from its phylogeographic landscape and human migration.

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.

Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain cluster.

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.

Insight into Population Structure and Drug Resistance of Pediatric Tuberculosis Strains from China and Russia Gained through Whole-Genome Sequencing.

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.

Impact of pathobiological diversity of Mycobacterium tuberculosis on clinical features and lethal outcome of tuberculosis.

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.

Mycobacterium bovis and M. caprae in Bulgaria: insight into transmission and phylogeography gained through whole-genome sequencing.

BACKGROUND: This study aimed to characterize recent Mycobacterium bovis/M. caprae isolates from Bulgaria by whole-genome sequencing (WGS) to gain a first insight into their molecular diversity, transmission, and position within the global phylogeography of this important zoonotic species. RESULTS: The isolates were obtained from cattle in diverse locations of Bulgaria in 2015-2020 and were identified by microbiological and PCR assays. WGS data were used for phylogenetic analysis that also included M. bovis global dataset. Thirty-seven M. bovis/caprae isolates from Bulgaria were studied and 34 of them were SNP genotyped. The isolates were subdivided into 3 major phylogenetic groups. Type Mbovis-13 (Eu2 complex [western Europe and northern Africa]) included one isolate. Mbovis-37 type included 5 isolates outside of known clonal complexes. The Bulgarian M. caprae isolates formed a sub-group within the Mcaprae-27B cluster which also included 22 M. caprae isolates from Poland, Spain, Germany, and the Republic of Congo. The Bulgarian M. caprae isolates share their latest common ancestors with Spanish isolates. The Mbovis-37 group shares a distant common ancestor (pairwise distance 22-29 SNPs) with an isolate from Poland but was very distant (> 200 SNPs) from the rest of the tree. The Mbovis-13 group shares a common ancestor with two human isolates from Germany. Phylogeographically, both M. bovis clades had limited circulation in northeastern Bulgaria while the majority of the studied isolates (M. caprae) were from central and western provinces. A phylogenetic network-based analysis demonstrated that 11 Bulgarian isolates were separated by 1 to 6 SNPs within four clusters, mostly forming pairs of isolates. CONCLUSION: The obtained WGS analysis positioned the Bulgarian isolates within the global phylogeography of M. bovis/M. caprae. Hypothetically, the observed phylogenetic diversity may not have resulted from livestock trade routes, but instead may reflect the deeply rooted M. bovis/M. caprae phylogeography of Europe. A high level of genetic divergence between the majority of the studied isolates suggests limited active transmission of bTB in Bulgaria during the survey period. At the same time, a possibility of the endemic presence of circulating bTB strains in the form of the latent persistent disease cannot be ruled out.

First insight into the whole-genome sequence variations in Mycobacterium bovis BCG-1 (Russia) vaccine seed lots and their progeny clinical isolates from children with BCG-induced adverse events.

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.

Mycobacterium tuberculosis RD-Rio Strain in Kazakhstan.

Mycobacterium tuberculosis RD-Rio strains are still rare in the former Soviet Union countries and Asia. We describe a strain in Kazakhstan that belongs to the RD-Rio secondary branch, which is endemic to northwest Russia and eastern Europe. Although RD-Rio strains are frequently multidrug resistant, this heterogeneous branch included only drug-susceptible isolates.

Simple Assay for Detection of the Central Asia Outbreak Clade of the Mycobacterium tuberculosis Beijing Genotype.

The Central Asia outbreak (CAO) clade is a branch of the Mycobacterium tuberculosis Beijing genotype that is associated with multidrug resistance, increased transmissibility, and epidemic spread in parts of the former Soviet Union. Furthermore, migration flows bring these strains far beyond their areas of origin. We aimed to find a specific molecular marker of the Beijing CAO clade and develop a simple and affordable method for its detection. Based on the bioinformatics analysis of the large M. tuberculosis whole-genome sequencing (WGS) data set (n = 1,398), we identified an IS6110 insertion in the Rv1359-Rv1360 intergenic region as a specific molecular marker of the CAO clade. We further designed and optimized a multiplex PCR method to detect this insertion. The method was validated in silico with the recently published WGS data set from Central Asia (n = 277) and experimentally with M. tuberculosis isolates from European and Asian parts of Russia, the former Soviet Union, and East Asia (n = 319). The developed molecular assay may be recommended for rapid screening of retrospective collections and for prospective surveillance when comprehensive but expensive WGS is not available or practical. The assay may be especially useful in high multidrug-resistant tuberculosis (MDR-TB) burden countries of the former Soviet Union and in countries with respective immigrant communities.

The role of IS6110 in micro- and macroevolution of Mycobacterium tuberculosis lineage 2.

The insertion sequence 6110 (IS6110) is the most studied transposable element in the Mycobacterium tuberculosis complex species. The element plays a significant role in genome plasticity of this important human pathogen, but still many causes and consequences of its transposition have not been fully studied. Here, we analyzed insertion sites for 902 Mycobacterium tuberculosis lineage 2 strains using whole-genome sequencing data. In total, 17,972 insertions were found, corresponding to 827 independent positions in the genome of the reference strain H37Rv. To trace the history of IS6110 expansion since proto-Beijing strains up to modern sublineages, we looked at the distribution of IS6110 across the genome-wide SNP-based phylogenetic tree. This analysis demonstrated a stepwise transposition of IS6110 that occurs by «copy-and-paste¼ mechanism. Additionally, we detected evolutionary-scale and sublineage-specific integration sites, which can be used for typing and for understanding the reasons for the success of the lineage. A significant part of such insertions affected the genes that are essential for the pathogen. Finally, we identified and confirmed deletions that occurred between differently oriented elements, which is uncommon for this family of insertion elements and appears to be another mechanism of genome variability.

New epidemic cluster of pre-extensively drug resistant isolates of Mycobacterium tuberculosis Ural family emerging in Eastern Europe.

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.

Whole-Genome Analysis of Mycobacterium tuberculosis from Patients with Tuberculous Spondylitis, Russia.

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.

Emerging resistant clones of Mycobacterium tuberculosis in a spatiotemporal context.

Objectives: We assessed the genetic structure of the Mycobacterium tuberculosis population in Estonia with a special focus on major epidemic/endemic clones and drug resistance determinants. We investigated the hypothesis of the decisive impact of massive human influx on the locally circulating genotypes. Estonia received a mass immigration from Russia during 1945-90 followed by enhanced interaction with the EU since 1991. Methods: The study sample included M. tuberculosis isolates from patients newly diagnosed with TB in 2014 in North Estonia (including the capital Tallinn). The isolates were subjected to first- and second-line drug susceptibility testing, detection of mutations in rpoB, katG, inhA, rrs, embB and gyrA and lineage/clone-specific genotyping. Results: Of the M. tuberculosis isolates, 39.8% were assigned to the Beijing genotype; 56.8% of them were MDR. In contrast, all three major non-Beijing genotypes (LAM, Haarlem and Ural) were mainly drug susceptible. MDR was more prevalent among Beijing B0/W148-cluster isolates (81.8%) compared with other Beijing isolates (20.0%; P = 0.0007). The pre-XDR phenotype was found in eight isolates, of which six belonged to Beijing B0/W148. All rifampicin-resistant and ofloxacin-resistant and 97% of isoniazid-resistant isolates harboured resistance mutations in rpoB, gyrA and katG. The rpoB S531L, katG S315T and embB M306V mutations were the most prevalent. Conclusions: The major pool of the Beijing isolates was brought to Estonia before 1990. However, an active circulation of the most hazardous MDR-associated Beijing B0/W148-cluster started only in the last 20 years and its significantly increased circulation presents the major threat to TB control in Estonia. The overwhelming prevalence of the rpoB531 and katG315 mutations in the MDR-associated Beijing isolates requires attention.

Emerging peak on the phylogeographic landscape of Mycobacterium tuberculosis in West Asia: Definitely smoke, likely fire.

To date, a major attention was justly given to the global lineages of Mycobacterium tuberculosis. Here, we demonstrated an importance of the minor ones, on an example of intriguing and underestimated NEW-1 family that belongs to Euro-American lineage (lineage 4). Analysis of the global WGS/NGS datasets (5715 strains) identified 2235 strains of Lineage 4 and 66 strains of sublineage L4.5. This latter is marked with RD122 genomic deletion and includes NEW-1 family. Phylogenomic analysis confirmed a separate position of the NEW-1 family that we tentatively designate L4.5.1/Iran. We propose an evolution/migration scenario starting with origin of L4.5 1000-1300 ya in China, subsequent origin of the pre-NEW-1 intermediate genotype in Tibet, further migration to Xinjiang/Uyghur, and finally to Iran since 800 ya (origin of NEW-1), possibly, via expansion of the Mongol Yuan empire. Analysis of longitudinal phylogeographic datasets revealed a sharp increase in prevalence of NEW-1 strains in Iran and its eastwards neighbors in the last 20years; most alarmingly, it is accompanied with significant association with multidrug resistance (MDR). Ongoing migration, especially, Afghan refugees flows to developed countries emphasize a risk of the wider spread of the epidemic MDR subtype within NEW-1 family that we coin as emerging resistant clone of M. tuberculosis in West Asia.

Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China.

Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains.

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis.

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

Latin-American-Mediterranean lineage of Mycobacterium tuberculosis: Human traces across pathogen's phylogeography.

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

Tuberculous spondylitis in Russia and prominent role of multidrug-resistant clone Mycobacterium tuberculosis Beijing B0/W148.

Extrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance of Mycobacterium tuberculosis isolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study of M. tuberculosis isolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n = 80); the other main families were T (n = 11), Ural (n = 7), and LAM (n = 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%; P < 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (n = 7). A notable prevalence of the rpoB531 and katG315 mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.

Trends in molecular epidemiology of drug-resistant tuberculosis in Republic of Karelia, Russian Federation.

BACKGROUND: Russian Republic of Karelia is located at the Russian-Finnish border. It contains most of the historical Karelia land inhabited with autochthonous Karels and more recently migrated Russians. Although tuberculosis (TB) incidence in Karelia is decreasing, it remains high (45.8/100 000 in 2014) with the rate of multi-drug resistance (MDR) among newly diagnosed TB patients reaching 46.5 %. The study aimed to genetically characterize Mycobacterium tuberculosis isolates obtained at different time points from TB patients from Karelia to gain insight into the phylogeographic specificity of the circulating genotypes and to assess trends in evolution of drug resistant subpopulations. METHODS: The sample included 150 M. tuberculosis isolates: 78 isolated in 2013-2014 ("new" collection) and 72 isolated in 2006 ("old" collection). Drug susceptibility testing was done by the method of absolute concentrations. Spoligotyping was used to test genotype-specific markers of a Latin-American-Mediterranean (LAM) family and its sublineages as well as a Beijing B0/W148-cluster. RESULTS: The largest spoligotypes were SIT1 (Beijing family, n = 42) and SIT40 (T family, n = 5). Beijing family was the largest (n = 43) followed by T (n = 11), Ural (n = 10) and LAM (n = 8). Successful Russian clone, Beijing В0/W148, was identified in 15 (34.9 %) of 43 Beijing isolates; all В0/W148 isolates were drug-resistant. Seven of 8 LAM isolates belonged to the RD115/LAM-RUS branch, 1 - to the LAM RD174/RD-Rio sublineage. MDR was found in Beijing (32/43), Ural (3/10), and LAM (3/8). In contrast, all T isolates were pansusceptible. Comparison of drug resistant subgroups of the new and old collections showed an increasing prevalence of the B0/W148 clonal cluster, from 18.0 % (mainly polyresistant) in 2006 to 32.6 % in 2014 (mainly MDR and pre-XDR). The West-east increasing gradient is observed for the Ural genotype that may be defined a 'Russian' strain. In contrast, the spoligotype SIT40 of the T family appears to be a historical Karelian strain. CONCLUSIONS: Circulation of the MDR M. tuberculosis isolates of the Beijing genotype and its B0/W148 cluster continues to critically influence the current situation with the MDR-TB control in northwestern Russia including the Republic of Karelia. Revealed phylogeographic patterns of some genotypes reflect a complex demographic history of Karelia within the course of the 20(th) century.

Insights into the origin, emergence, and current spread of a successful Russian clone of Mycobacterium tuberculosis.

Mycobacterium tuberculosis variant Beijing B0/W148 is regarded as a successful clone of M. tuberculosis that is widespread in the former Soviet Union and respective immigrant communities. Understanding the pathobiology and phylogeography of this notorious strain may help to clarify its origin and evolutionary history and the driving forces behind its emergence and current dissemination. I present the first review and analysis of all available data on the subject. In spite of the common perception of the omnipresence of B0/W148 across post-Soviet countries, its geographic distribution shows a peculiar clinal gradient. Its frequency peaks in Siberian Russia and, to a lesser extent, in the European part of the former Soviet Union. In contrast, the frequency of B0/W148 is sharply decreased in the Asian part of the former Soviet Union, and it is absent in autochthonous populations elsewhere in the world. Placing the molecular, clinical, and epidemiological features in a broad historical, demographic, and ecological context, I put forward two interdependent hypotheses. First, B0/W148 likely originated in Siberia, and its primary dispersal was driven by a massive population outflow from Siberia to European Russia in the 1960s to 1980s. Second, a historically recent, phylogenetically demonstrated successful dissemination of the Beijing B0/W148 strain was triggered by the advent and wide use of modern antituberculosis (anti-TB) drugs and was due to the remarkable capacity of this strain to acquire drug resistance. In contrast, there is some indication, but not yet systematic proof, of an enhanced virulence of this strain.

Mycobacterium tuberculosis Latin American-Mediterranean family and its sublineages in the light of robust evolutionary markers.

Mycobacterium tuberculosis has a clonal population structure, and the Latin American-Mediterranean (LAM) family is one of the largest and most widespread within this species, showing evidence for remarkable pathobiology and a confusing phylogeny. Here, we applied robust phylogenetic markers to study the evolution of the LAM family and its major sublineages circulating in Russia and neighboring countries. A total of 250 M. tuberculosis isolates were confirmed to belong to the LAM family based on the analysis of the LAM-specific single-nucleotide polymorphisms (SNPs) in the Rv3062 and Rv0129c genes. At this stage, the family status was rectified for 121 isolates misleadingly assigned by CRISPR spoligotyping to non-LAM families (T1- or T5-RUS1). Consequently, the reestimated LAM prevalence rate increased 2-fold in Russia and Kazakhstan and 4-fold in Belarus. The majority (91.8 to 98.7%) of the LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, the Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. Taken together, our findings and further analyses suggest a monophyletic origin of LAM-RUS: at a historically distant time, in Russia, in a small founding bacterial/human population. Its dissemination pattern and high prevalence rate in Northern Eurasia may indicate a long-term coexistence of the LAM-RUS sublineage and local human populations hypothetically leading to coadaptation and reduced pathogenicity of the relatively more ancient clones, such as spoligotype international type 254 (SIT254), compared to the more recent SIT252 and SIT266 clones. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The spread of RD-Rio strains is not as global as commonly claimed and is determined largely by human migration flows (rather than by pathobiological properties of these strains). Consequently, a host population factor appears to play a major role in shaping the in situ dissemination pattern of the imported strains in an autochthonous population.