A multifaceted interplay between virulence, drug resistance, and the phylogeographic landscape of Mycobacterium tuberculosis.

Latin-American Mediterranean (LAM) family is one of the most significant and global genotypes of Mycobacterium tuberculosis. Here, we used the murine model to study the virulence and lethality of the genetically and epidemiologically distinct LAM strains. The pathobiological characteristics of the four LAM strains (three drug resistant and one drug susceptible) and the susceptible reference strain H37Rv were studied in the C57BL/6 mouse model. The whole-genome sequencing was performed using the HiSeq Illumina platform, followed by bioinformatics and phylogenetic analysis. The susceptible strain H37Rv showed the highest virulence. Drug-susceptible LAM strain (spoligotype SIT264) was more virulent than three multidrug-resistant (MDR) strains (SIT252, SIT254, and SIT266). All three MDR isolates were low lethal, while the susceptible isolate and H37Rv were moderately/highly lethal. Putting the genomic, phenotypic, and virulence features of the LAM strains/spoligotypes in the context of their dynamic phylogeography over 20 years reveals three types of relationships between virulence, resistance, and transmission. First, the most virulent and more lethal drug-susceptible SIT264 increased its circulation in parts of Russia. Second, moderately virulent and pre-XDR SIT266 was prevalent in Belarus and continues to be visible in North-West Russia. Third, the low virulent and MDR strain SIT252 previously considered as emerging has disappeared from the population. These findings suggest that strain virulence impacts the transmission, irrespective of drug resistance properties. The increasing circulation of susceptible but more virulent and lethal strains implies that personalized TB treatment should consider not only resistance but also the virulence of the infecting M. tuberculosis strains. IMPORTANCE The study is multidisciplinary and investigates the epidemically/clinically important and global lineage of Mycobacterium tuberculosis, named Latin-American-Mediterranean (LAM), yet insufficiently studied with regard to its pathobiology. We studied different LAM strains (epidemic vs endemic and resistant vs susceptible) in the murine model and using whole-genome analysis. We also collected long-term, 20-year data on their prevalence in Eurasia. The findings are both expected and unexpected. (i) We observe that a drug-susceptible but highly virulent strain increased its prevalence. (ii) By contrast, the multidrug-resistant (MDR) but low-virulent, low-lethal strain (that we considered as emerging 15 years ago) has almost disappeared. (iii) Finally, an intermediate case is the MDR strain with moderate virulence that continues to circulate. We conclude that (i) the former and latter strains are the most hazardous and require close epidemiological monitoring, and (ii) personalized TB treatment should consider not only drug resistance but also the virulence of the infecting strains and development of anti-virulence drugs 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.

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.

Genetic Diversity and Primary Drug Resistance of Mycobacterium tuberculosis Beijing Genotype Strains in Northwestern Russia.

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.

Molecular Insight into Mycobacterium tuberculosis Resistance to Nitrofuranyl Amides Gained through Metagenomics-like Analysis of Spontaneous Mutants.

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.

Population structure of Mycobacterium tuberculosis from referral clinics in Western Siberia, Russia: Before and during the Covid-19 pandemic.

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.

Molecular snapshot of drug-resistant Mycobacterium tuberculosis strains from the Plateau State, Nigeria.

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.

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.

Practical approach to detection and surveillance of emerging highly resistant Mycobacterium tuberculosis Beijing 1071-32-cluster.

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.

Extremely lethal and hypervirulent Mycobacterium tuberculosis strain cluster emerging in Far East, Russia.

Mycobacterium tuberculosis strains of the early ancient sublineage of the Beijing genotype are mostly drug susceptible and mainly circulate in East Asia. We have recently discovered two clusters of this sublineage emerging in the Asian part of Russia (VNTR-defined 1071-32 and 14717-15 types) and, to our surprise, both were strongly MDR/XDR-associated. Here, we evaluated their pathogenic features. The clinical isolates and reference laboratory strain H37Rv were investigated in the C57BL/6 mouse model to assess their virulence and lethality properties. The BACTEC MGIT 960 system was used to study the in vitro growth characteristics. In the murine model, strains 396 (14717-15-cluster, from Buryatia, Far East) and 6691 (1071-32-cluster, from Omsk, Siberia) demonstrated contrasting properties. The 396-infected group had significantly higher mortality, more weight loss, higher bacterial burden, and more severe lung pathology. Furthermore, compared to the previously published data on other Russian epidemic Beijing strains (B0/W148, CAO, Central Asian Russian), strain 396 demonstrated the highest mortality. Under the in vitro growth experiment, cluster 14717-15 isolates had significantly shorter lag-phase. To conclude, low-virulent MDR strain 6691 belongs to the Beijing 1071-32-cluster widespread across FSU countries but at low prevalence. This corresponds to common expectation that multiple drug resistance mutations reduce fitness and virulence. In contrast, highly lethal and hypervirulent MDR strain 396 represents an intriguing Beijing 14717-15 cluster predominant only in Buryatia, Far East (16%), sporadically found beyond it, but not forming clusters of transmission. Further in-depth study of this most virulent Russian Beijing cluster is warranted.

Spatiotemporal dynamics of drug-resistant Mycobacterium tuberculosis: Contrasting trends and implications for tuberculosis control in EU high-priority country.

Different and contrasting trends related to human migration and the implementation of health control programmes influence the spread of drug-resistant tuberculosis (TB). We analysed the Mycobacterium tuberculosis population structure in Estonia, a high-priority EU country for TB control, to detect the dynamic changes and underlying factors. The study collection included 278 M. tuberculosis isolates recovered in 1999 and 2014-2015. The isolates were subjected to drug susceptibility testing, genotyping and analysis of sublineage/cluster-specific markers and drug resistance mutations. The Beijing genotype was the most prevalent, and its rate increased from 28.6% in 1999 to 38.5% in 2015 (p = .09). The non-Beijing strains represented Euro-American lineage (Latin American Mediterranean [LAM], Ural, Haarlem, T, X genotypes) and Indo-Oceanic lineage (one EAI-IND isolate). The proportion of isolates resistant to two or more drugs increased from 22.4% to 29.1% (p = .1). The pre-XDR/XDR isolates were identified only within the Beijing genotype. In contrast, the drug resistance rate decreased in the LAM genotype from 42.1% to 11.8% (p = .05). The Beijing B0/W148-cluster ('successful Russian strain') included only MDR, pre-XDR or XDR isolates. All B0/W148-cluster isolates were resistant to two or more drugs compared to 28% of the Beijing 94-32-cluster (p = .0002). The Beijing genotype was not identified in the isolates from patients born in Estonia before 1940 compared to its 35.2% rate among other patients. In summary, the circulation of the highly drug-resistant isolates of the Beijing B0/W148 subtype, the increased prevalence of the Beijing genotype among HIV-coinfected patients and the increased number of patients with alcohol abuse (47.5%) present major challenges of the current TB control in Estonia. The Beijing genotype was likely brought to Estonia after 1945 due to the massive human influx from the Soviet Union. In contrast, the main genotypes of the Euro-American lineage were likely endemic in Estonia during all 20th century.

Genetic Variation Putatively Associated with Mycobacterium tuberculosis Resistance to Perchlozone, a New Thiosemicarbazone: Clues from Whole Genome Sequencing and Implications for Treatment of Multidrug-Resistant Tuberculosis.

Perchlozone ([PCZ] 4-thioureido-iminomethylpyridinium perchlorate) is a new thiosemicarbazone approved for the treatment of multidrug-resistant tuberculosis (MDR-TB) in Russia and some other countries. The ethA and hadABC mutations may confer PCZ resistance. At the same time, ethA mutations are known to mediate resistance to ethionamide (ETH) and prothionamide (PTH). We aimed to study the genetic variation underlying Mycobacterium tuberculosis resistance to PCZ through whole genome sequencing (WGS) of consecutive isolates recovered during long-term treatment. This prospective study included patients admitted in 2018-2019 to the regional tuberculosis dispensary, Kaliningrad, Russia, whose treatment regimen included PCZ. Multiple M. tuberculosis isolates were recovered during PCZ treatment, and the bacterial DNA was subjected to WGS followed by bioinformatics analysis. We identified mutations in the genes putatively associated with PCZ resistance, ethA, and hadA. The most frequent one was a frameshift ethA 106 GA > G (seven of nine patients) and most of the other mutations were also likely present before PCZ treatment. In one patient, a frameshift mutation ethA 702 CT > C emerged after six months of PCZ treatment. A frequent presence of cross-resistance mutations to PCZ and ETH/PTH should be taken into consideration when PCZ is included in the treatment regimen of MDR-TB patients.

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.

Increased transmissibility of Russian successful strain Beijing B0/W148 of Mycobacterium tuberculosis: Indirect clues from history and demographics.

The local situation with tuberculosis (TB) is shaped by the complex interplay of multiple factors related to both human host and Mycobacterium tuberculosis. We hypothesized that TB epidemiology in the rural regions in the Soviet Union was impacted by construction of the Gulag camps and significant incoming migration. This molecular M. tuberculosis study was conducted in 2017 in the Komi Republic in northern Russia, a region with high rate (26%) of primary multidrug-resistant (MDR) TB. MDR was detected in 30.8% (40/130) isolates; eight were extensively drug resistant. The Beijing genotype was predominant (56.2%). The main Beijing subtypes B0/W148 and 94-32 differed in the MDR rate, 83.3% and 27.2%, respectively. The non-Beijing isolates represented five genotypes (LAM, Ural, Haarlem, X, T). The proportion of Beijing B0/W148 in the "camp" cities (originated from Gulag camps) was twice as large as in other districts of the Komi Republic. To conclude, сirculation of the MDR-associated Beijing B0/W148 cluster critically influences the current situation with MDR-TB in this Russian region. The increased prevalence of B0/W148 in the urban setting on the whole, and in the "camp cities", in particular, indirectly points to the increased transmission capacity of this successful Russian strain of M. tuberculosis.

Genomic signatures of drug resistance in highly resistant Mycobacterium tuberculosis strains of the early ancient sublineage of Beijing genotype in Russia.

The Mycobacterium tuberculosis Beijing genotype is a clinically and epidemiologically important lineage that is subdivided into ancient/ancestral and modern strains. In our previous study in western Siberia, we identified variable number of tandem repeats (VNTR)-based clusters within the early ancient sublineage of the Beijing genotype characterized by an unexpectedly high rate of extensive drug resistance (XDR). In the current study, next generation sequencing data were analysed to gain insight into genomic signatures underlying drug resistance of these strains. A total of 184 genomes of the Beijing early ancient sublineage from Russia (16), China (15), Japan (36), Korea (25), Vietnam (18), Thailand (73), and the USA (1) were used for phylogenetic analysis. The drug-resistant profile was deduced genotypically. The Russian isolates were distributed into two clusters and were all drug resistant, mainly pre-XDR and XDR. The largest of these clusters included only Russian isolates from remote locations in both Asian and European parts of the country. All its isolates had a quadruple drug resistance (to isoniazid, rifampin, ethambutol and streptomycin) due to the 6-mutation signature (KatG Ser315Thr, KatG Ile335Val, RpoB Ser450Leu, RpoC Asp485Asn, EmbB Gln497Arg, and RpsL Lys43Arg). In most samples, it was complemented with additional and different pncA, gyrA and rrs mutations leading to the pre-XDR/XDR genotype. Phylogenomic analysis indicates a distant origin of this Russian resistant cluster in the early 1970s but location and circumstances are yet to be clarified.

First insight into phylogeography of Mycobacterium bovis and M. caprae from cattle in Bulgaria.

Bovine tuberculosis (bTB) represents a significant economic burden to the agriculture. In spite of decades of the control program, Mycobacterium bovis infection levels in cattle in Bulgaria continued to rise over recent years. In order to gain a better understanding of the M. bovis diversity, we used spoligotyping for strain differentiation and the data were compared to the international databases Mbovis.org and SITVIT2 for shared type and clade assignment. Study sample included 30 M. tuberculosis complex isolates from cattle originating from different regions of Bulgaria. The isolates were subdivided by spoligotyping into 4 spoligotypes: 2 types shared by 20 and 8 isolates and 2 singletons. SITVIT2-defined types SIT645 and SIT647 belonged to the common and classical bovine ecotype M. bovis (9 isolates) while types SIT120 and SIT339 belonged to the M. caprae ecotype (21 isolates). A certain phylogeographic gradient of the spoligotypes and clades at the within-country level was observed: M. caprae was prevalent in the central/southwestern, while classical M. bovis in the northeastern Bulgaria. Whereas all four types have global or European circulation, neither was described in the neighboring Balkan countries. M. caprae isolates identified in this study mostly belong to the Central/Eastern European cluster. In summary, this study provided a first insight into phylogeography of M. bovis in Bulgaria and described, for the first time, M. caprae as an important infectious agent of bTB in this country.

Molecular characteristics of Mycobacterium tuberculosis in the "closed" Russian town with limited population migration.

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.

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.