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PubMed | University of Latvia, St Petersburg Pasteur Institute, Ural Research Institute of Phthisiopulmonology, Aitkhozhin Institute of Molecular Biology and Biochemistry and 5 more.
Type: | Journal: Molecular phylogenetics and evolution | Year: 2016

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.


News Article | August 22, 2016
Site: www.rdmag.com

Researchers from the Federal Research and Clinical Centre of Physical-Chemical Medicine, and staff from MIPT's Systems Biology Laboratory, the Research Institute of Phthisiopulmonology and the St. Petersburg Pasteur Institute, conducted a large-scale analysis of the proteins and genomes of mycobacterium tuberculosis strains that are common in Russia and countries of the former Soviet Union and found features that provide a possible explanation for their epidemiological success. A paper detailing the results has been published in the prestigious journal Scientific Reports, part of Nature Publishing Group. Up until the 20th century, tuberculosis was considered an incurable disease and, despite newly developed methods of curing it at its early stages, the death rate is still high. There are 22 countries, including Russia, in which the infection rate is four times greater than in the rest of the world. It is important to note that the term "tuberculosis" covers a wide range of bacteria strains that cause the disease. Strains of the Beijing family (this genotype was first discovered in Beijing) are prevalent in Russia - every year about 150,000 people are infected with it. To understand the reason behind the "success" of this strain, scientists compared proteins produced by Beijing B0/W148 with a control strain. In order to do this, separated bacterial proteins were enzymatically cleaved into smaller fragments - peptides and their mass and relative abundance were measured precisely using mass spectrometry. After analysing the data collected, the scientists knew which and how many proteins there were in each strain. It was found that in Beijing B0/W148 strains, 266 proteins were differentially abundant compared with the control strain. 57 of them were entirely absent in the study group and 17, on the contrary, were unique to it, others differed quantitatively. Analysis of the functions associated with differing proteins revealed that in Beijing B0/W148 strains there are more proteins producing long-chain fatty acids and less proteins destroying them. Bacteria use these acids to produce mycolic acids, which integrate themselves in the bacterial cell membrane and make it waxy, which is why mycobacteria can survive and even reproduce in macrophages (special human cells that destroy foreign substances). Normally, if a bacterium is "eaten" by a macrophage it dies. However, mycobacterium tuberculosis strains have evolved to reproduce in macrophages and in doing so they hide from the immune system. Mycolic acids not only protect bacteria, but also play a crucial role in synthesizing substances that inhibit macrophages so they stop fighting disease. The features of lipid metabolism that have been discovered could explain the success of Beijing B0/W148 strains in relation to other tuberculosis mycobacteria.


Mokrousov I.,St Petersburg Pasteur Institute | Narvskaya O.,St Petersburg Pasteur Institute | Vyazovaya A.,St Petersburg Pasteur Institute | Otten T.,Research Institute of Phthisiopulmonology | And 5 more authors.
Journal of Clinical Microbiology | Year: 2012

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. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Mokrousov I.,St Petersburg Pasteur Institute | Vyazovaya A.,St Petersburg Pasteur Institute | Otten T.,Research Institute of Phthisiopulmonology | Zhuravlev V.,Research Institute of Phthisiopulmonology | And 5 more authors.
PLoS ONE | Year: 2012

This study aimed to characterize the population structure of Mycobacterium tuberculosis in Pskov oblast in northwestern Russia, to view it in the geographical context, to compare drug resistance properties across major genetic families. Ninety M. tuberculosis strains from tuberculosis (TB) patients, permanent residents in Pskov oblast were subjected to LAM-specific IS6110-PCR and spoligotyping, followed by comparison with SITVITWEB and MIRU-VNTRplus databases. The Beijing genotype (n = 40) was found the most prevalent followed by LAM (n = 18), T (n = 13), Haarlem (n = 10), Ural (n = 5), and Manu2 (n = 1); the family status remained unknown for 3 isolates. The high rate of Beijing genotype and prevalence of LAM family are similar to those in the other Russian settings. A feature specific for M. tuberculosis population in Pskov is a relatively higher rate of Haarlem and T types. Beijing strains were further typed with 12-MIRU (followed by comparison with proprietary global database) and 3 hypervariable loci QUB-3232, VNTR-3820, VNTR-4120. The 12-MIRU typing differentiated 40 Beijing strains into 14 types (HGI = 0.82) while two largest types were M2 (223325153533) prevalent throughout former USSR and M11 (223325173533) prevalent in Russia and East Asia. The use of 3 hypervariable loci increased a discrimination of the Beijing strains (18 profiles, HGI = 0.89). Both major families Beijing and LAM had similar rate of MDR strains (62.5 and 55.6%, respectively) that was significantly higher than in other strains (21.9%; P = 0.001 and 0.03, respectively). The rpoB531 mutations were more frequently found in Beijing strains while LAM drug resistant strains mainly harbored rpoB516 and inhA -15 mutations. Taken together with a high rate of multidrug resistance among Beijing strains from new TB cases (79.3% versus 44.4% in LAM), these findings suggest the critical impact of the Beijing genotype on the current situation with MDR-TB in the Pskov region in northwestern Russia. © 2012 Mokrousov et al.


PubMed | Instituto Adolfo Lutz, University Hospital Clementino Fraga Filho, St Petersburg Pasteur Institute, University of Campinas and 3 more.
Type: | Journal: Tuberculosis (Edinburgh, Scotland) | Year: 2015

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.To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17).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.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.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.


Vyazovaya A.,St Petersburg Pasteur Institute | Mokrousov I.,St Petersburg Pasteur Institute | Solovieva N.,Research Institute of Phthisiopulmonology | Mushkin A.,Research Institute of Phthisiopulmonology | And 4 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2015

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 tandemrepeat (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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Mokrousov I.,St Petersburg Pasteur Institute | Vyazovaya A.,St Petersburg Pasteur Institute | Zhuravlev V.,Research Institute of Phthisiopulmonology | Otten T.,Research Institute of Phthisiopulmonology | And 6 more authors.
Journal of Clinical Microbiology | Year: 2014

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. © 2014, American Society for Microbiology. All Rights Reserved.


Zimenkov D.V.,RAS Engelhardt Institute of Molecular Biology | Kulagina E.V.,RAS Engelhardt Institute of Molecular Biology | Antonova O.V.,RAS Engelhardt Institute of Molecular Biology | Zhuravlev V.Y.,Research Institute of Phthisiopulmonology | Gryadunov D.A.,RAS Engelhardt Institute of Molecular Biology
Journal of Antimicrobial Chemotherapy | Year: 2016

Background: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. Methods: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. Results: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. Conclusions: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex. © The Author 2016.


PubMed | Research Institute of Phthisiopulmonology and St Petersburg Pasteur Institute
Type: Journal Article | Journal: Antimicrobial agents and chemotherapy | Year: 2015

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.


PubMed | Research Institute of Phthisiopulmonology and RAS Engelhardt Institute of Molecular Biology
Type: Journal Article | Journal: The Journal of antimicrobial chemotherapy | Year: 2016

Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs.We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray.The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype.The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.

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