Annotation of the results obtained in 2021
In 2021, 1 application was filed and 1 more patent application was prepared for methods for detecting resistant clusters. The results are presented at 4 international congresses. Two articles of 2021 are reflected in the RSF News https://www.rscf.ru/news/biology/shtamm-vozbuditelya-tuberkuleza-obnaruzhennyy-na-dalnem-vostoke-okazalsya-vysokoletalnym/; https://rscf.ru/news/biology/lekarstvenno-ustoychivyy-shtamm/ The aim of the study is a comprehensive analysis of the biological properties of new highly resistant and potentially epidemic genotypes of Mycobacterium tuberculosis to assess their virulence, resistance, evolutionary history and prognosis for the future. We would like to emphasize the multi-vector nature of the project with the logical interconnection of all its components. In 2021 (the third final year of the project), studies were carried out simultaneously in several interrelated areas (phylogeographic, phylogenetic, bioinformatics analysis, insilico modeling, analysis of pathobiological properties in a mouse model of strains of different genotypes and comparison with clinical significance in relation to the human population), Combining the results of which made it possible to obtain a holistic picture of the evolution and epidemiology of significant M. tuberculosis genotypes and to formulate new tasks for the application for the continuation of the project. In 2021, research was carried out in the following areas, in which results of world priority were obtained. - Genome-wide and phylogenetic analysis of strains of ancient Beijing sublines to identify phylogenetic neutral SNP markers of the resistant Russian cluster 14717-15 and to determine SNPs leading to significant amino acid substitutions based on in silico analysis, primarily in genes of virulence, adaptation, cell wall synthesis. Assembly and annotation of genomes of two strains of this cluster and their deposition. - Development of a method for rapid PCR detection Beijing 14717-15, screening of retrospective and new DNA collections and analysis of the global genomic database of the Beijing genotype for their identification. - Study of virulence and lethality in a mouse model taken as a comparison of strains of another significant genetic family LAM (Latin American Mediterranean). - Assessment of the clinical significance of epidemic and endemic genotypes in relation to the human population based on the analysis of the outcome of the disease (diagnosis and mortality). PHILOGENETIC ANALYSIS OF CLUSTERS 1071-32 AND 14717-15 WITHIN THE FRAMEWORK OF GLOBAL PHILOGENY BASED ON ANALYSIS OF ADDITIONAL GENOMS As a result of sequencing in 2021 an additional sample of 19 strains of the ancient Beijing subline, a new dendrogram of the Russian and global sample of strains of the early ancient Beijing subline (224 genomes) from Russia, China, Japan, Korea, Vietnam and Thailand was built. Strains from Russia fell into two distinct clusters in different branches of the tree (FIG 2). All Russian strains were genotypically multidrug resistant (at least), and due to additional resistance mutations, many (especially in cluster 1071-32) were pre-XDR (widely drug resistant). The largest of these clusters 1071–32 (Fig. 3) included only Russian isolates from European and Asian Russia. All were resistant to INH, RIF, STR, EMB due to 6 mutations in KatG315 / 335, RpoB450, RpoC485, EmbB497, RpsL43. Most had mutations in pncA, gyrA, rrs, eis, that is, they were pre-XDR. Based on the number of SNPs separating the most distant isolates of this cluster and based on an estimate of the rate of mutations in the M. tuberculois genome (0.5 SNP / genome / year), we assume its origin in the 1970s. The strains from Thailand were relatively closest neighbors of these strains. Taking into account the isolation of the Omsk region (where the largest percentage of this cluster is in Russia), such a relationship looks strange and requires additional research. The Russian isolates of the smaller cluster 14717-15 were from Asian Russia (Fig. 4). They shared two mutations in resistance rpsL Lys88Arg katG Srr315Thr. Phylogenetically, the closest neighbors were isolates from Korea, while Russian isolates from both Omsk and Buryatia and one of the Korean isolates had a characteristic spoligoprofile SIT269 (derived from the classic spoligoprofile Beijing - SIT1). This makes a plausible hypothesis about the Korean origin of this cluster, or rather its Russian founder strain. SELECTION OF SNP-MARKERS FOR GENOTYPE BEIJING 14717-15, DEVELOPMENT AND VALIDATION OF THE METHOD OF ITS DETECTION, AND SCREENING OF COLLECTIONS Analysis of genomic variability unique for individual isolates and their clusters revealed 40 specific single nucleotide substitutions (SNPs) specific for the highly lethal and highly virulent cluster of Beijing 14717-15 strains. Among these SNPs, we chose a neutral substitution at the genomic position 1448330 G> T i.e. in the gene Rv1293 303G> T, codon 101GCG / GCT (Ala / Ala). A neutral mutation is not subject to the risk of natural selection and, given the improbability of reversion, such a mutation is especially suitable for use as a specific marker for gene diagnostics. Analysis of the gene region in which this mutation is located revealed that as a result of it, a recognition site for the restriction endonuclease HhaI GCGC arises. This makes it possible to detect this mutation on the basis of PCR amplification of a gene fragment followed by treatment with restriction enzyme HhaI and separation of restriction products in an agarose gel. A PATENT APPLICATION HAS BEEN PREPARED, which will be sent to Rospatent in December 2021. Additionally, we checked c using the PCR-RFLP method one more SNP in the genomic position 2423040 A> G (Rv2161c Val (s) 33Ala), which also has an extremely high PAM1 value, i.e. phylogenetically neutral. For its detection, restriction with the same HhaI restriction enzyme was used. The use of two SNPs increases the reliability of the detection of a named cluster. Both PCR-RFLP schemes were tested and optimized for PCR conditions on isolates with known genome-wide sequences and VNTR profiles. Both mutations were found only in the strains of the Beijing 14717-15 cluster. Next, we screened geographic collections of isolates from various Russian regions for the presence of strains of this cluster. The method for detecting strains of the Beijing 14717-15 cluster based on PCR-RFLP analysis of two specific SNPs was applied to a collection of DNA isolates from different regions of Siberia and the Far East. Additionally, the identified phylogenetic SNPs of the Beijing 14717-15 cluster were screened in the global Beijing genome database, which includes more than 6000 genomes (fastq files). This database was compiled from genomic data extracted from GenBank / ENA in our collaboration with Dr. Joao Perdigao, Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa (Portugal). COMPARATIVE PHILOGEOGRAPHY. Comparison of the geography of subtype 14717-15 with the distribution of another studied subtype Beijing 1071-32 shows a greater distribution of the latter, but in a small proportion, with the exception of the Omsk region. A detailed analysis of the phylogeography of the Beijing 1071-32 cluster was presented in the 2020 report. In general, according to VNTR analysis, isolates from the Beijing 1071-32 cluster were mainly detected in Russia with a clearly increasing prevalence in Omsk, Siberia, but these isolates were also present in other regions of Russia and the countries of the former Soviet Union in Central Asia and the Caucasus. Outside the former Soviet Union, Beijing 1071-32 isolates have been described in Serbia (Merker et al., 2015), Albania (Tafaj et al., 2020), and Greece (Ioannidis et al., 2017). These are Balkan countries, but it is not known if this fact already reflects the local circulation of this strain, and not an independent import from the former USSR. DNA from Albania and Greece were available for this study and we confirmed that they contain all 9 mutations, i.e. 3 neutral SNPs and 6 resistance mutations. Three isolates from Greece were obtained from immigrants from the former USSR, namely from Georgia, and two others from patients from Greece. The Peking genotype is extremely rare in Albania, and it has previously been suggested that these isolates in Albania may be related to neighboring Greece. A large European multicenter study of drug-resistant tuberculosis in the EU identified Beijing 1071-32 isolates a decade ago and designated them as the European resistant cluster ECDC_10 (de Beer et al., 2014). This further highlights the presence of the Beijing 1071-32 MDR strain in the EU and the relevance of tracking them on a more global scale, not limited to Russia and other countries of the former Soviet Union. ASSESSMENT OF VIRULENCE OF LATIN AMERICAN MEDITERRANEAN (LAM) GENOTYPE STRAINS IN A MOUSE MODEL. Comparison with data for epidemic clusters of the Beijing genotype. Of the 4 studied strains (spoligotypes), two were previously identified as emergent, highly resistant and potentially epidemic, SIT266 - in Belarus and SIT252 - in the European part of Russia. At the same time, two other spoligotypes (SIT264, SIT254) are not marked with special pathobiological properties. Below is a table with information on strains and spoligotypes. When comparing the virulence of clinical isolates of the genetic family Latin American Mediterranean (LAM) 7074, 3929, 306, and 4542 and the reference strain M. tuberculosis H37Rv, it can be argued that the intravenous administration of all studied clinical strains causes generalized tuberculosis in infected mice with predominant lung involvement. According to the totality of indicators of the severity of the course of the tuberculosis process (survival rate, body weight dynamics, biometric indicators, the degree of prevalence of foci of specific inflammation in the lungs and the level of MBT contamination of the lungs and spleen), the virulence of all clinical strains of M. tuberculosis (7074, 3929, 306 and 4542) was significantly below the virulence of the reference strain M. tuberculosis H37Rv. The minimum virulence was recorded in three low-lethal strains LAM 7074, 3929 and 4542 (all MDR), moderate in strain 306 (sensitive). Comparison of the results for LAM strains with previously studied strains of the ancient and modern subline of the Beijing genotype shows a clear connection between the success of the epidemic Russian Beijing clusters with their increased virulence and lethality. For a further in-depth understanding of the results obtained at the molecular level of both the host organism and the microorganism, it is necessary to study by methods of systems biology. This is planned in our application for a 2-year extension of the grant. INFLUENCE OF PATHOBIOLOGICAL DIVERSITY OF MYCOBACTERIUM TUBERCULOSIS ON CLINICAL MANIFESTATIONS AND MORTALITY OF TUBERCULOSIS The infamous Beijing genotype prevails in the population of Mycobacterium tuberculosis in Russia, the main epidemic and endemic variants of which are characterized by the opposite properties of drug resistance and virulence. We examined how these characteristics of the strain might affect the progression of pulmonary tuberculosis (TB) in terms of clinical manifestations and death. The research collection included 548 M. tuberculosis isolates isolated in 2013–2019. from 548 patients with newly diagnosed pulmonary tuberculosis in Omsk, Western Siberia, Russia. The strains were subjected to drug susceptibility testing and genotyping to identify clones, sublines and subtypes (within the Beijing genotype). The Beijing genotype was identified in 370 (67.5%) strains studied. The strongest association with MDR was found for the Beijing B0 / W148 epidemic cluster (modern subline) and two recently discovered clusters 1071-32 and 14717-15 of the ancient Beijing subline. The group of patients infected with the hypervirulent and highly lethal (in a mouse model) cluster Beijing 14717-15 showed the highest mortality rate (58.3%) compared to Beijing B0 / W148 (31.4%; P = 0.06). Beijing Central Asian / Russian (29.7%, P = 0.037) and non-Beijing strains (15.2%, P = 0.001). Cluster 14717-15 mainly included isolates from patients with infiltrative, but not with fibrocavernous and disseminated tuberculosis. On the contrary, the group infected with the cluster with low virulence and MDR 1071-32 had the highest level of fibrocavernous TB, which probably reflects the ability of these strains for long-term survival and chronic course of the TB process. Subgroups of patients infected with the modern subline Beijing and the ancient cluster 14717-15 were dominated by patients aged <44 years, which probably correlates with the active ongoing transmission of these strains. In conclusion, this study emphasizes that not only drug resistance, but also the virulence of strains must be taken into account when introducing personalized medicine and TB treatment.

 

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Annotation of the results obtained in 2019
ABBREVIATIONS. MIC - minimal inhibitory concentration, MDR-multiple drug resistance, XDR-extensive drug resistance, SNP-single nucleotide polymorphism, NGS-next generation sequencing, WGS-whole genome sequencing. Genomic analysis of early ancient Beijing strains and determination of cluster-specific mutations. The initial collection of genomes (fastq files) was extracted from GenBank, Broad Institute database, GMTV database and included 3089 samples, in particular 521 genomes from East and Southeast Asia (Japan, China, Korea, Vietnam, Thailand), and 1067 Russian samples. Of these, 117 genomes were assigned to the early ancient sublineage with the wild-type mutT4-48 codon. They represented the following countries and regions: 27 - Korea, 20 - Russia, 28 - China, 36 - Japan, 1 - USA, 5 - of unknown origin. The phylogenetic analysis of SNPs covering the entire genome subdivided all genomes of the early ancient sublineage of Beijing into 10 monophyletic clades: 5 clades belonged to the subgroup with intact RD181, and the other 5 clades belonged to the branch with deleted RD181. 24-VNTR and spoligotyping profiles were obtained for Russian strains, and this information was used to correlate previously identified VNTR clusters of the early ancient Beijing with clades based on NGS. In addition, in silico spoligotyping was done for all genomes included in the tree. This showed that most isolates had the Beijing SIT1 profile (signals 35 to 43), while 4 isolates had the SIT269 spoligoprofile (signals 37 to 43). For a large branch on the tree, including only Russian multidrug-resistant strains of the 1071-32 cluster, cluster-specific SNPs were determined (excluding resistance genes). Summing up, there were 15 SNPs in the intergenic regions, 10 in the PE_PGRS and PPE genes (1 nonsense, 6 missense, including two with a significant amino acid substitution, and 3 synonymous mutations), in other genes - 96, including - synonymous - 40, non-synonymous - 54, nonsense - 2. According to the functional category, 96 mutations were identified in the coding regions. Of these, for mutations leading to significant changes in amino acids (significance index 3 and 4), the genes were in the following categories: Intermediate metabolism and respiration - 6, Processes associated with the cell and cell wall - 4, Conservative hypothetical proteins - 2, Lipid metabolism - 1. A method for detecting a highly-resistant cluster (1071-32-cluster of early ancient Beijing) in real-time PCR format. (this part of the workplan was completed earlier than planned) The choice was made among synonymous mutations with a zero significance index, which seem to be the most phylogenetically neutral. Based on the identified SNPs specific for a high-resistance cluster, a method for detecting this cluster in real-time PCR format using LNA-modified probes was developed. The method is in the process of (i) evaluating three such SNPs to select the most optimal and (ii) subsequent validation on local collections. The patent application is scheduled for the 1 quarter of 2020. Characterization of Mycobacterium tuberculosis populations from Russian regions and from other countries to identify strains of the ancient sublineage of the Beijing genotype. Characterization of the Ural region M. tuberculosis collection In the studied sample, males were 87 (79.8%), females - 22 (20.2%). Genotyping revealed the predominance of isolates of the Beijing genotype both among newly diagnosed patients 75.8% (50/66) and previously treated patients 83.7% (36/43). The Beijing genotype also dominated the groups of HIV-positive and HIV-negative tuberculosis patients. Beijing B0 / W148 and Beijing 94-32 were the predominant clusters and were isolated in 50.0% (33/66) and 22.7% (15/66) of newly diagnosed patients, respectively. Genotyping of Beijing isolates at two MIRU-VNTR loci: MIRU26 and QUB26 identified nine groups. The largest of them included 39 isolates of the Beijing B0/W148 group and was characterized by seven repeats at both loci. Interestingly, 11 isolates of Beijing B0/W148 were characterized by unusual (for this cluster) two repeats at the QUB26 locus (designated for brevity as “QUB26 / 2”). Most isolates of the Beijing 94-32 cluster were characterized by five repeats in MIRU26 and eight repeats in QUB26. Spoligotyping of non-Beijing isolates subdivided them into nine SITs belonging to four genetic families. Of these, SIT35 / Ural was the largest cluster and included 9 (8.3%) isolates. Spoligotypes SIT 262 / Ural; SIT 42, SIT252, SIT254 / LAM; SIT334 / T1; SIT182 / Haarlem; SIT402 / Unknown was represented by one or two isolates. Among HIV-positive patients, only SIT35 / Ural was detected in 4 (9.5%) cases. The Beijing B0/W148 cluster comprised only MDR, XDR, and multiresistant isolates. Isolates of Beijing B0/W148 QUB26/2 were further investigated for resistance mutations. All 11 isolates had a similar mutation profile associated with resistance to isoniazid (katGS315T), rifampicin (rpoBS531L), ethambutol (embBM306V) and aminoglycosides (eis-g10a). Mutations of resistance to fluoroquinolones were also detected in three isolates, namely: gyrAD94G, gyrAA90V and gyrBI525L. Non-Beijing isolates were mostly susceptible to anti-TB drugs. MDR isolates were found only in the Ural family of SIT35 and SIT262, but SIT35 also included sensitive isolates. Only one XDR isolate (SIT252 / LAM) was found among non-Beijing isolates. Conclusions. In this study, we characterized the population structure of M. tuberculosis in the city of Novouralsk. Genotyping revealed the dominance of isolates of the most notorious variant of M. tuberculosis in Russia, the Beijing B0/W148 cluster, which accounts for a high percentage of MDR-TB cases among new cases of tuberculosis. Characterization of the M. tuberculosis population in Buryatia: an unusual structure with the presence of an early ancient Beijing cluster. 381 M. tuberculosis strains were genotyped at 24 MIRU-VNTR loci and regions RD 105, 207 and 181. The Beijing genotype made up the majority of strains in the studied sample (64.1% 216/337 in 2010-2014 and 75% in the sample of 44 isolates in 2019). Strains without deletions in RD 105/207 were identified as LAM - 16.9% (57/337); Ural - 7.4% (25/337); T-3.9% (13/337). The total proportion of the most common Beijing subtypes – 94-32-cluster and B0/W148, was lower than in the Irkutsk region and Sakha (Yakutia) (Zhdanova S., et al., 2017). These two subtypes accounted for a total of 56.0% of Beijing strains (121/216), which is not characteristic of the structure of the MBT population circulating in Russia (Mokrousov I., 2012; 2015). The described structure of Beijing strains is different from other territories due to the significant presence of other pathogen variants characteristic of Buryatia. In the study sample, the subtype Beijing MIT642 (early ancient sublineage of Beijing) was found in 15.4% (52/337). This subtype was found among isolates obtained from newly diagnosed TB patients (3.4% - 24/186), and in previously treated patients (7.0% - 28/147). Moreover, the frequency of its detection among patients with primary and chronic TB did not significantly differ (χ2 = 0.2.35; p = 0.125). The ethnicity of the patients with tuberculosis caused by the Beijing MIT 642 subtype did not matter: there were no significant differences in the frequency of detection in the group of Slavs (20/159) and Buryats (8/70) (χ2 = 0.001 with Yats correction, p> 0.05) . Such a uniform distribution of strains of the subtype Beijing MIT 642, may indicate its long-standing circulation, which allowed it to spread in both ethnic groups. The profiles of Beijing MIT 642, which were present only in isolated cases in collections from other regions of Russia (Mokrousov I. et al., 2015), can be considered endemic for Transbaikalia (Buryatia and Transbaikal Territory). The presence of a common reservoir of infection for different ethnic groups is also determined by the common clusters of MIRU-VNTR-24 profiles of Beijing MIT 642 strains. A high level of clustering (CR = 0.73) was detected along with the discovery of two large groups with identical profiles 224233342644425173343732 (24 strains) and 224233342644425173343832 (13 strains), equally often distributed among different ethnic components of the cohort of TB patients from Buryatia. Moreover, all profiles at 24 MIRU-VNTR loci were assigned to the BL7 clonal complex (Merker M. et al., 2015), that includes ancestral (= ancient) sublineage of the Beijing genotype. Molecular Characteristics of the M. tuberculosis collection in Estonia The study collection was M. tuberculosis strains obtained from 161 newly diagnosed tuberculosis patients in 1999. The 1999 collection was analyzed to assess the dynamic changes and will be supplemented by an analysis of the recent collection. As a result of genotyping, 28.6% (46 of 161) of M. tuberculosis strains were assigned to the Beijing genotype. The Beijing B0/W148 and 94-32 clusters were identified in 13.7% (22/161) and 8.7% (14/161) cases; respectively. Of the 161 strains, 106 (65.8%) were drug susceptible, MDR was detected in 23 (14.8%) strains. Multiple drug resistance (MDR) was detected in 78.3% (18/23) of the Beijing genotype strains and in 21.7% (5/23) of strains of other genetic families. MDR strains were dominated by strains of the B0/W148 cluster (68.2%, 15/22) than other Beijing strains (13.0%, 3/23, P = 0.0006). In 115 non-Beijing strains, 31 spoligotypes (SIT) of different genetic lineages were identified: T (24.2%), Haarlem (17.4%), Ural (14.3%), LAM (11.8%), X ( 0.6%). Four isolates had new spoligotypes not found in SITVIT_WEB. One isolate (SIT11) was assigned to EAI3-IND. M. tuberculosis population structure in Albania: place and role of Russian epidemic genotypes Albania is a Balkan country with a moderate / low incidence of tuberculosis (TB) and a very low prevalence of drug-resistant TB. We analyzed the nationwide multi-year collection of Mycobacterium tuberculosis to identify possible dynamic trends in the development of tuberculosis in Albania with an emphasis on drug resistance and endemic/epidemic clones including originating from countries of the former USSR. After World War II, Albania had close links with the Soviet Union in 1948–1960 and with China in the 1960–1970s and had limited ties with other countries until the 1990s. We hypothesized that such a demographic history could affect the population structure of M. tuberculosis in Albania. 745 isolates collected in 2007–2011 were divided into 107 spoligotypes and 353 MIRU types. Based on the phylogenetic analysis of MIRU-VNTR, isolates were assigned to the following lineages/families: Lineage 2 (5 Beijing isolates), Lineage 3 (1 CAS-Delhi) isolate), and Lineage 4 (Cameroon, Uganda, Ghana; NEW-1-related ; Ural, Haarlem, LAM, S, TUR; and unclassified isolates). Most isolates (454/745) were intermediately located on the global VNTR tree and were not grouped with any reference profile; they were remotely related to the Lineage 4 families, and we defined them as “unclassified L4 isolates.” A significantly higher proportion of drug resistance was observed for (i) the Beijing genotype compared to all other isolates (60%, P = 0.008), (ii) “unclassified L4” compared to all other isolates (13.9%, P = 0 , 04) and (iii) SIT2936 compared with other “unclassified L4” (34.3%, P = 0.0006). Most of the LAM (33/49) and Beijing (3/5) belonged to the VNTR types characteristic of Russia and the countries of the former Soviet Union Based on the 24-MIRU-VNTR clustering, 5 isolates were assigned to the Beijing genotype. Three strains had specific Mlva profiles that have attracted attention in recent years. One susceptible strain was of the type Mlva 94-32 and, thus, belonged to the Central Asian-Russian clade, which is widespread in the countries of the former USSR and associated with MDR in Central Asia. Two other Beijing strains belonged to the Mlva 1071-32 cluster, which was recently described as a highly resistant genotype in Russia within the early ancient subline of Beijing (Mokrousov et al., 2019). This type was also described in a previous Albanian study (Tafaj et al., 2009), in Greece (Ioannidis et al., 2017), and in Serbia (Merker et al., 2015). However, two of the three Albanian strains were drug-sensitive. Given the specificity of types Mlva 94-32 and 1071-32 for Russia and the former USSR, these isolates could have entered Albania either recently with migrants from the countries of the former USSR or from the Soviet Union in an earlier period. Albanian isolates were mostly sensitive and therefore could not come from MDR-associated isolates currently circulating in the countries of the former USSR and among migrants of the former USSR in Greece. On the contrary, in 1948-1960, there was close interaction and exchange between Albania and the Soviet Union when thousands of Albanians studied in the Soviet Union (Krisafi, 2015). Accordingly, it seems plausible that these strains were imported to Albania 70-80 years ago, and circulated under the same public health conditions as the local pool of strains, and, like local strains, remained largely susceptible to antibiotics. Our results highlight the specific nature of the M. tuberculosis population in Albania, which is dominated by local and unclassified genotypes in Lineage 4, as well as European genotypes and epidemiologically significant clones originating from countries of the former Soviet Union. At the same time, these imported clones remain drug-sensitive. Analysis of the Beijing genotype in Central America: a possible link with the countries of the former USSR or East Asia The aim of the study was to characterize the most common cluster of the Beijing genotype in the province of Colon (Panama). The DNA of 42 M. tuberculosis isolates (Colon, Panama, 2018) was genotyped at 24 MIRU-VNTR loci (plus 4 hypervariable loci) and genome sequencing using the MiSeq platform. A cluster-specific PCR targeting the common Beijing strain was used prospectively to identify new cases in a sample of 26 cases. For comparison, we used the global collection of NGS (> 9000 genomes) and the MIRU-VNTR database (> 8000 isolates [Mokrousov, 2015; Merker et al., 2015]). The Panama dominant cluster within the Beijing genotype belonged to the modern sublineage of the Beijing genotype (NTF :: IS6110, mutation in ogt12) and its Asian-African branch 3, an endemic and less common genotype in East Asia, also described in southern Africa. Based on 24-MIRU-VNTR typing and comparison with databases, the Panama strains of the Beijing genotype were assigned to three closely related Mlva types (MIRU-VNTRplus): 19 isolates - type 1048-33, 2 isolates - type 1048-32, 1 isolate - type 342-33. Compared with the global 24-MIRU Beijing sample (4987 isolates) (Merker et al., 2015, Mokrousov, 2015), these three types were present in countries of East Asia and southern Africa. PARTICIPATION IN CONFERENCES: 2 plenary lectures: 5th International congress of Moroccan Association of Microbiology (Ifrane, Morocco); VIII Congress of the National Association of Phthisiatrists (St. Petersburg). 1 oral report: International Workshop "Next generation sequencing and bioinformatics tools for Mycobacterium tuberculosis drug resistance detection and epidemiological analysis" (St. Petersburg). 2 poster presentations: 40th Annual Congress of European Society of Mycobacteriology (Valencia, Spain); 50 World Conference on Lung Health (Hyderabad, India). ARTICLES Umpeleva T, Belousova K, Golubeva L, Boteva T, Morozova I, Vyazovaya A, Mokrousov I, Eremeeva N, Vakhrusheva D. Molecular characteristics of Mycobacterium tuberculosis in the “closed” Russian town with limited population migration // Infection Genetics and Evolution. Under Minor revision. Tafaj S, Mokrousov I, Borroni E, Trovato A et al. Peculiar features of the Mycobacterium tuberculosis population structure in Albania // Infection Genetics and Evolution. https://doi.org/10.1016/j.meegid.2019.104136 In press.

 

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Annotation of the results obtained in 2020
The aim of the study is a comprehensive analysis of the biological properties of the emerging highly resistant and potentially epidemic genotypes of Mycobacterium tuberculosis to assess their virulence, resistance, evolutionary history and prognosis for the future. We would like to emphasize the multi-vector nature of the project with the logical interconnection of all its components. In 2020, research was carried out in the following major areas. - primary characterization of new regional collections by genotyping methods (VNTR, spoligotyping) to determine the structure of the entire population at the level of families, sublineage, genotypes and subtypes, including Russian clusters of early ancient Beijing 1071-32 and 14717-15. - Genome-wide and phylogenetic analysis of strains of ancient Beijing sublineages (1) to identify phylogenetically neutral SNP markers of a large resistant Russian cluster 1071-32 and (2) to determine SNPs leading to significant amino acid substitutions based on in silico analysis, primarily in virulence, adaptation, cell wall synthesis genes. Assembly and annotation of genomes of strains of two clusters and their deposition. - Development of a method for Real Time PCR detection of Beijing 1071-32 and screening of retrospective DNA collections and analysis of the global genomic database of the Beijing genotype to identify Beijing 1071-32. Incorporation of new genomic data into a more comprehensive phylogenetic analysis to estimate the timing of origin of cluster 1071-32. - Study of the growth rate of strains Beijing 1071-32 and 14717-15 in an in vitro model - Study of virulence and lethality of Beijing strains 1071-32 and 14717-15 in a mouse model. Phylogenetic analysis of a global sample of strains of the early ancient sublineage of the Beijing genotype from Russia, East and Southeast Asia localized Russian isolates into two monophyletic clusters with 100% bootstrap support, which corresponded to clusters based on VNTR typing, designated as Mlva 1071-32 and 14717 -15, according to the international nomenclature MIRU-VNTRplus.org. These clusters belonged to the early ancient Beijing sublineages 1 (intact locus RD181) and 2 (del_RD181), respectively. All Russian isolates were multiply- or widely drug-resistant (MDR/XDR) and had a certain phylogeographic affinity: cluster 1071-32 was detected in strains from the European part of Russia and Western Siberia, while cluster 14717-15 - in strains from Western and, mainly Eastern Siberia. For the larger Russian cluster Beijing 1071-32, cluster-specific SNPs were identified, of which 96 were in coding sequences. Based on the assessment of the significance indices, 14 mutations were identified, leading to hypothetically significant amino acid substitutions. Further, the influence of the most significant mutations on protein structures was assessed using in silico structural analysis and on the basis of an assessment of the electrostatic potential around the mutated amino acids. As a result, no significant effect on structure and function was found for most of the studied amino acid substitutions. In addition to the genetic polymorphisms leading to the functionally significant mutations described above, phylogenetic SNPs were also identified for the Beijing 1071-32 cluster, i.e., neutral mutations with zero amino acid substitution significance. Based on two such SNPs, a method was developed for detecting strains Beijing 1071-32 in real-time PCR format using LNA probes. The method was optimized for DNA strains with available genomic data and known alleles in target genes/positions. Further, the method was used to screen retrospective and new DNA collections from various regions of the Russian Federation and a number of countries in Eastern Europe, Asia, Africa and South America for the possible identification of strains of this cluster. The preparation of a patent application is pending, so we do not provide details on the exact positions of the SNP data and the structure of primers and probes. The identified phylogenetic SNPs of the Beijing 1071-32 cluster were screened in the global Beijing genome database, which includes more than 6000 genomes (fastq files). As a result of the analysis, additional genomes of strains of cluster 1071-32 were identified - strains from the countries of the former USSR (Azerbaijan, Georgia, Tajikistan), as well as from China, which may be ancestral in relation to this cluster. These genomes were included in additional analysis to estimate the time of origin of the 1071-32 resistant cluster. Significantly greater than expected divergence of strains from FSU countries was found. This result will be rechecked and we plan to repeat all stages of bioinformatics processing of files, starting with fastq/vcf files and up to building a minimal network. The developed RT-PCR method for the detection of the Beijing 1071-32 cluster based on 2 snips was used to screen new and archived DNA collections from different regions of Russia (Ural, Siberia, Northwest) and foreign ones. Preliminary results show the Russian specificity of this cluster, while its identification in single isolates in Albania and Greece is most likely associated with immigrants from the countries of the former USSR (isolates in Greece) and possibly movements between neighboring Balkan countries (between northern Greece and Albania). Also, the 100% specificity and sensitivity of this method for the detection of the Beijing 1071-32 cluster was confirmed. The genomes of 2 strains of cluster 1071-32 were assembled, annotated and deposited in GenBank. Short reads of the strains were collected de novo, by the collector Bandage v. 0.8.1. Genome coverage was 31.9 x. The resulting contigs were annotated using the Prokaryotic Genome Annotation Pipeline (PGAP). For strain 2044, the total genome length was 4,339,788 bp. The genome contains 4088 genes, of which 4037 are coding (CDSs), 3898 are protein-coding genes, 51 are RNA genes, 139 are pseudogenes. Among the 51 RNA genes there are 45 tRNA genes, 3 non-coding RNAs (ncRNAs). In parallel with the above analysis, members of the research group in Yekaterinburg, Omsk and Irkutsk collected new collections of M. tuberculosis isolates and performed their basic characteristics for further analysis and identification of strains of the Beijing genotype, its ancient sublineages and resistant clusters 1071-32 and 14717-15. Simultaneously with the genomic and phylogenetic analysis described above, studies were carried out to study the biological properties of two Russian resistant clusters in in vivo and in vitro models. In vivo model: investigation of the virulence of clinical isolates in modeling tuberculosis infection in C57BL/6 mice. Based on the results of an assessment of the virulence of clinical MDR isolates 396y (Beijing 14717-15 cluster, Buryatia) and 6691 (cluster 1071-32, Omsk) in comparison with the reference M. tuberculosis strain H37Rv, we suggest that both studied clinical strains are capable of causing intravenous infection of mice with a tuberculous process with a predominant lesion of the lungs. The presence of a correlation between the mortality rates of infected mice with one or another MBT strain and the severity of pathological changes in the lung tissue, as well as the bacterial load in the lungs and spleen, was shown. In terms of the totality of indicators of the severity of the course of the tuberculosis process (survival rate, body weight dynamics, biometric indicators, the prevalence of specific inflammation foci in the lungs and the level of MBT contamination of the lungs and spleen), the virulence of the clinical M. tuberculosis 6691 strain was significantly lower than the virulence of the standardized M. tuberculosis H37Rv strain. In contrast the virulent properties of the clinical strain 396y were more pronounced than those of M. tuberculosis strain H37Rv. First of all, this was recorded by the dynamics of the lethality of mice, according to which the mass death of mice, as well as 100% lethality, were recorded earlier in the group of animals infected with the 396y strain. The results of the assessment of M. tuberculosis infection of the lungs, which is considered an integral indicator of the severity of the course of infection, are in agreement with these data, since the level of bacterial load of the MBT lungs at almost all periods of observation of an already developed disease (except for the last examination period) was significantly higher when using strain 396y. In addition, when mice were infected with M. tuberculosis 396y, the maximum values of biometric parameters, like almost all characteristics of the prevalence of pathological changes in the lungs, were recorded earlier than in other groups. In vitro model: evaluation of differences in the growth rate of strains of two resistant clusters of the ancient subline of the Beijing genotype. The results of studying the growth parameters of the strains of the two clusters 1071-32 and 14717-15 show within each group a significant diversity in the values of growth parameters. Comparison of the studied groups showed that some differences between them were only in two growth parameters - the duration of the lag phase and the point of maximum increase in fluorescence (p = 0.05). The strains of the 1071-32 cluster more slowly exited the lag phases and at a later date had the maximum increase in fluorescence. However, according to the integral growth parameter - the coefficient r, characterizing the features of the MBT growth curve as a whole, according to the U criterion, the groups did not differ from each other, with a weak tendency of the strains of the 1071-32 cluster to slow growth. THUS, Beijing 1071-32 is less virulent and lethal than the highly lethal Beijing 14717-15, which mainly circulates in Buryatia. At the same time, using the example of 1071-32, we see that the presence of numerous resistance mutations can indeed lead to a decrease in the survival (fitness) and transmissibility of the strain as a whole, since this cluster makes up an insignificant share of the population in most regions (except for the Omsk region), although it is quite widespread. For example, we can note its identification in other countries, not only in the countries of the former USSR, but (most likely due to migrants from the former USSR) - in Albania and Greece. Comparison of models for direct and indirect analysis of virulence in vivo and in vitro shows that the growth rate indicator for assessing pathogenicity and transmission is not sufficiently informative. On the contrary, the mouse model of virulence revealed significant differences between the studied clusters and identified the Buryat cluster 14717-15 as highly virulent and highly lethal, and more lethal than the well-known epidemic genotypes in Russia and Central Asia Beijing B0 / W148 and Beijing CAO. These highly resistant genetic clusters reflect two paradigms of evolution and adaptation. Comparison with the available data on other genotypes allows us to formulate a hypothesis about the relationship between virulence and drug resistance as the main factor in the spread of pathogenic respiratory bacteria.

 

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