Genetic analysis of Mycobacterium tuberculosis strains isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping

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1 INT J TUBERC LUNG DIS 9(7): The Union Genetic analysis of Mycobacterium tuberculosis strains isolated in Ural region, Russian Federation, by MIRU-VNTR genotyping S. Y. Kovalev,* E. Y. Kamaev, M. A. Kravchenko, N. E. Kurepina, S. N. Skorniakov * Ural State Aleksei Maksimovich Gorky University, Ekaterinburg, Ural Research Institute for Phthisiopulmonology, Ekaterinburg, Russian Federation; Public Health Research Institute (PHRI) TB Center, International Center for Public Health, Newark, New Jersey, USA SUMMARY SETTING: The Ural region in Russia is one of the areas most affected by a high incidence of tuberculosis (TB). Molecular epidemiological studies able to trace Mycobacterium tuberculosis transmission are of particular significance. OBJECTIVE: To characterize the population of M. tuberculosis strains circulating in the Ural region, to detect the predominant genotypes and to evaluate their phylogenetic relationship and epidemiological significance. DESIGN: Ninety-two M. tuberculosis clinical samples originating from the Ural region were genotyped using the MIRU-VNTR method. RESULTS: Two major phylogenetically distinct groups of isolates were identified: the W-Beijing family (54.3%) and a previously unreported cluster, named the Ural group (15.2%). Forty-seven different MIRU profiles were identified, including 38 unique (41.3%) and 54 isolates grouped into nine clusters (from 2 to 28 isolates in each cluster). Genetic diversity within the clusters was shown by additional sub-typing of M. tuberculosis isolates in nine additional QUB-VNTR loci. CONCLUSION: W-Beijing family isolates are associated with multiresistance to anti-tuberculosis drugs. It is possible that the strains of this family play a significant role in the spread of multidrug-resistant TB over the Ural region. KEY WORDS: Mycobacterium tuberculosis; molecular epidemiology; MIRU-VNTR genotyping; drug resistance; W-Beijing family ACCORDING TO World Health Organization (WHO) data, the tuberculosis (TB) epidemic remains a serious problem worldwide. The Ural region in Russia is one of the most affected areas, with an incidence of TB in 2003 of 74.3 cases per population. 1 Under these circumstances, molecular epidemiological studies able to trace Mycobacterium tuberculosis transmission are of particular significance. Genotyping methods are based on the analysis of genomic polymorphism of M. tuberculosis strains isolated from TB patients from different regions. 2,3 Some of the established genotyping techniques are either laborious or expensive (IS6110 restriction fragment length polymorphism [RFLP] genotyping) or do not possess enough discriminatory power (spoligotyping), and require uniform data processing systems, complicating the comparison of results from different laboratories due to subjectivity in the analysis. Genotyping of M. tuberculosis strains based on the polymorphism of 12 selected variable-number tandem repeat (VNTR) loci, referred to as mycobacterial interspersed repetitive units (MIRUs), allows rapid analysis of the results (numbers of repeats) as digital patterns, with subsequent comparison with worldwide databases. 4 6 MIRU-VNTR typing allows fast, objective, high resolution monitoring of epidemiological situations throughout the country. MIRU-VNTR typing was recently implemented in the Russian Federation to characterize the M. tuberculosis population. 7,8 The aim of this study was to characterize the population of M. tuberculosis strains circulating in the Ural region, Russian Federation, to detect the predominant genotypes and to evaluate their phylogenetic relationship and epidemiological significance. MATERIALS AND METHODS M. tuberculosis strains Ninety-eight M. tuberculosis isolates (convenience sample) obtained from October 2001 to June 2002 from 92 patients (25 females and 67 males, aged 17 71) with pulmonary TB (PTB) and with no known contacts were analyzed. Thirty-four of the samples were isolated from newly diagnosed patients. Three cultures were isolated from a single patient, and two cultures were isolated from each of four patients over Correspondence to: Dr Sergey Yu Kovalev, Ural State University, 51 Lenin Ave, Ekaterinburg , Russia. Tel: ( 7) Fax: ( 7) sergey.kovalev@usu.ru Article submitted 15 September Final version accepted 8 November 2004.

2 Genetic analysis of M. tuberculosis by MIRU-VNTR 747 an interval of 3 6 months. Laboratory strains M. tuberculosis H37Rv, H37Ra and M. bovis BCG were used as controls; their MIRU-VNTR profiles have been reported previously. 8 M. tuberculosis cultures were grown on Löwenstein-Jensen medium for 6 8 weeks. Susceptibility to rifampicin (RMP), isoniazid (INH), ethambutol, streptomycin, kanamycin, mycobutin, and cycloserine was determined by the indirect absolute concentration method. 9 The M. tuberculosis cultures were identified as multidrug-resistant (MDR) if they were resistant to at least RMP and INH. The identification of isolates as M. tuberculosis cultures was confirmed by multiplex polymerase chain reaction (PCR) amplification of IS6110, the 65-kDa antigen (Rv0440, groel2, TubercuList (Institut Pasteur, Paris, France), as well as spacers 33 and/or 34 in the direct repeat (DR) locus of the genome. 10 MIRU-VNTR genotyping DNA was isolated by incubation of M. tuberculosis cells resuspended in sterile TE buffer at 95 C for 30 min, with subsequent centrifugation at g; the supernatant was used directly for PCR-based analysis. MIRU-VNTR typing was carried out as described elsewhere. 11 The number of tandem repeats was determined according to the size of the PCR-amplified fragment electrophoresed in 1.8% agarose gel in 0.5x TBE buffer. The subtyping of clustered isolates was done with additional VNTR loci exact tandem repeats A and B (ETRA and ETRB), QUB11a, QUB11b, QUB18 and QUB26 by PCR amplification according to the conditions described elsewhere, 12 and QUB11451, QUB1895 and QUB Two or more isolates with identical MIRU-VNTR profiles were attributed to one cluster (SK1-SK9); isolates with identical QUB VNTR profiles in one cluster were attributed as subclusters (SK1-SK9 A-E). The isolate from the W-Beijing family was identified by the presence of an IS6110 insertion in the NTF locus by multiplex PCR. 14 PCR amplicon of 223 base pairs (bp) confirmed the presence of an IS6110 insertion in the NTF-1 position of this locus. IS6110 insertions in other positions of the locus (resulting in different sizes of PCR fragments) were ignored. Statistical analysis The discriminatory power of MIRU-VNTR typing was calculated using the Hunter-Gaston index. 15 Genetic distance was calculated as P distance or dissimilarity (D) distance value. Phylogenetic reconstruction was performed using the PAUP v.4.10b program 16 with the UPGMA (unweighted pair group method with arithmetic mean) algorithm. RESULTS MIRU-VNTR typing of M. tuberculosis clinical isolates Molecular analysis was conducted on 98 clinical isolates obtained from 92 patients with PTB. All 98 cultures were confirmed as belonging to the M. tuberculosis complex by differential multiplex PCR analysis. 10 MIRU profiles of six sequentially isolated cultures from five patients were found to be identical and were excluded from the analysis. The value of the Hunter-Gaston index for this set of isolates was 0.894, indicating that MIRU typing is capable of high discriminatory power. Analysis of allele polymorphism of 12 MIRU loci revealed the inherent difference in variability of number of repeats in each locus (Table 1). The allelic diversity of the MIRU loci varies widely, from 0.03 (loci 4 and 23) to 0.67 Table 1 The allele polymorphism of MIRU-VNTR loci of M. tuberculosis strains isolated from PTB patients in the Ural region Repeats MIRU loci n Index of allele polymorphism* * Index of allele polymorphism calculated according to the formula h 1 x i2 [n/(n 1)], where x i corresponds to the frequency of allele i in locus, and n corresponds to the number of isolates. MIRU-VNTR mycobacterial interspersed repetitive unit-variable number tandem repeats; PTB pulmonary tuberculosis.

3 748 The International Journal of Tuberculosis and Lung Disease (locus 26). Locus 24 is the exception, as it presents as a single copy in all isolates. Four MIRU loci (10, 26, 31 and 40) were found to be the most polymorphic, in agreement with results previously published. 17 The results of phylogenetic analysis of isolates are represented in the form of a dendrogram (Figure 1) which allows two major distinct evolutionary groups to be identified among 92 isolates. A comparison of MIRU-VNTR profiles obtained in this study with those available through the international databases (Institut Pasteur, Lille, France 18 and PHRI TB Center, Newark, NJ) has shown that the predominant group of isolates (n 50, 54.3%) belongs to the W-Beijing family (MIRU-VNTR profile ; invariable numbers of repeats are in bold). The second group has not been previously reported and has a characteristic profile (MIRU profile ). This group included 14 (15.2%) isolates and has been arbitrarily assigned as the Ural genotype. The Ural group of strains by MIRU profiles corresponds to the families of strains also known as KQ and LL (PHRI TB Center database of MIRU and RFLP profiles), previously isolated in Western Siberia, Russian Federation (N Kurepina, unpublished data). In addition to these two clonal families W-Beijing and Ural, five isolates were found to belong to the Haarlem family (MIRU-VNTR profile ) and one to the Africa family (MIRU profile ). 5 Eight isolates with the profiles were found to correspond to the MIRU-VNTR profiles of the AI strain family identified in the PHRI TB Center collection. The remaining 14 (15.2%) isolates could not be ascribed to any specific strain groups; they are therefore referred to as others for convenience of result interpretation (Figure 1). Characterization of identified MIRU clusters Within the 92 isolates analyzed, a total of 47 distinct MIRU profiles were detected; 38 (41.3%) were unique (detected in one isolated strain in this study), and 9 profiles comprised clusters containing 2 28 isolates each (n 54; 58.7%) (Figure 1). All clusters (with the exception of clusters SK4 and SK9) belonged to members of the W-Beijing family and/or the Ural group. Thus, 88% of isolates from the W-Beijing family (n 44; 4 clusters) and 43% of the Ural group (n 6; 3 clusters) were included in clusters, while the Haarlem and AI families each comprised small clusters with two isolates in each cluster. The Other group did not comprise clustered strains. Additional sub-typing of the clusters using two ETR and seven QUB-VNTR loci allowed for further differentiation Figure 1 Dendrogram of phylogenetic similarity of M. tuberculosis strains isolated in the Ural region according to the MIRU cluster analysis. k1-165 non-clustered isolates, SK1-9 MIRU clusters. MIRU mycobacterial interspersed repetitive unit.

4 Genetic analysis of M. tuberculosis by MIRU-VNTR 749 Figure 2 Dendrogram of the sub-clusters genetic similarity within MIRU-VNTR clusters of M. tuberculosis strains isolated in the Ural region. SK1-9 clusters; A, B, C sub-clusters. Bold lines indicate sub-clusters with IS6110 in chromosomal NTF region. Loci used for sub-clustering are listed as follows: ETRA, ETRB, QUB11a, QUB11b, QUB18, QUB26, QUB1451, QUB1895, QUB MIRU-VNTR mycobacterial interspersed repetitive unit-variable number tandem repeats; ETR exact tandem repeats. within clusters (Figure 2). We thus succeeded in separating the most numerous clusters SK1 (n 28) and SK2 (n 11) into 5 and 3 sub-clusters, respectively. Additional identification of the W-Beijing strains The relatedness of the clinical isolates to the W- Beijing strain family was confirmed by the presence of IS6110 in the NTF genome locus. 14 A PCR fragment of approximately 220 bp in length was identified in 49 isolates, including 46 strains from the W-Beijing family, two strains from the Others group and one strain from the Ural group. However, the 220-bp amplicon was not detected in strains from the sub-clusters SK1C (n 2), SK3A (n 1) or SK3B (n 1), determined as W-Beijing strains by MIRU-VNTR typing. Drug resistance of M. tuberculosis isolates Determination of drug susceptibility showed that 54 (58.4%) of 92 analyzed isolates were phenotypically resistant to one or more anti-tuberculosis drugs, including 23 (25.0%) isolates with MDR profiles. Cultures isolated from 58 (63.0%) patients previously treated with anti-tuberculosis drugs were found to be resistant to at least one drug in 65.5% (n 38) of the cases, while the cultures from 34 (37.0%) newly diagnosed patients were resistant in 44.1% (n 15) of the cases. Respectively 29.3% and 17.6% of the strains of these groups of patients were MDR. Table 2 shows the distribution of MIRU genotypes of clinical M. tuberculosis strains according to their drug resistance profiles. The strains attributed to the W-Beijing family (n 50) were resistant to one or more drugs in 70.0% (n 35) of cases. It should be noted that 19 (82.6%) of all 23 MDR strains belonged to the W-Beijing strain family. DISCUSSION The Russian Federation belongs to the group of countries with a high TB burden (85 cases per population in 2003). 19 Along with the high incidence rate, the problem is exacerbated by the constant increase of resistance to anti-tuberculosis drugs. 20 The emergence of drug-resistant strains is dangerously high in Russia. 21 In our study, 92 M. tuberculosis cultures (convenience sample) isolated from patients with PTB in the Ural region were analyzed using conventional typing

5 750 The International Journal of Tuberculosis and Lung Disease Table 2 Drug susceptibility of M. tuberculosis isolates from different MIRU groups and families MIRU genotype Drug-susceptible isolates n (%) Mono-resistant isolates n (%) Poly-resistant isolates (including MDR) MDR n (%) Beijing (n 50) 13 (26) 2 (4) 35 (70) 19 (38) Ural (n 14) 8 (57.1) 5 (35.7) 1 (7.1) 1 (7.1) Small clusters 7 (50) 0 7 (50) 3 (21.4) AI (n 8) Haarlem (n 5) Africa (n 1) Others (n 14) 10 (71.4) 4 (28.6) 0 0 Total (n 92) 38 (41.3) 11 (12.0) 43 (46.7) 23 (25.0) MIRU mycobacterial interspersed repetitive unit; MDR multidrug-resistant, defined as resistance to at least isoniazid and rifampicin. techniques (Figure 1). Two major phylogenetically distinct groups of strains dominate in the Ural region, and 92.6% of all clustered strains fall into these groups. Comparison with the international database shows that the most numerous group, consisting of 50 (54.3%) strains, belongs to the W-Beijing family. As previously published, isolates from the W-Beijing family represent the predominant group in different regions of the Russian Federation, including North-West Russia, 22 Central Russia 23 and Western Siberia. 24 It is possible that specific selective advantages (probably due to the particularity of their repair system 25 ) allow W- Beijing strains to successfully avoid the host immune response and succeed during the process of dissemination. A group of isolates (n 14, 15.2%) displaying an MIRU profile ( ) that has not previously been published, was identified and named the Ural family. The unique characteristic of this group includes 6 13 repeats in MIRU locus 10, and the presence of only one repeat in MIRU locus 26. The Ural genotype could represent an endemic group dispersed throughout the Russian Federation, as isolates with Ural MIRU genotype have also been collected from Western Siberia (PHRI TB Center collection). Sub-typing with two ETR and seven QUB-VNTR loci confirmed close relatedness of strains within clusters as well as within the separate families (Figure 2). The two largest clusters, SK1 (n 28) and SK2 (n 11), which belong to the W-Beijing family, were divided into 5 and 3 sub-clusters, respectively. Strains from sub-clusters SK1A (n 22) and SK2A (n 7) were analyzed for a total of 21 VNTR, and it was not possible to subdivide them further. However, another method of genotyping (IS6110 RFLP) might divide these sub-clusters into smaller groups. We suppose that clone expansion of the W-Beijing superclones SK1A and SK2A, demonstrated in our study, began relatively recently (before the beginning of the 1960s), as they consist of both drug-resistant and sensitive strains. It was recently shown for the Beijing strains isolated in North-West Russia 8 that MIRU VNTR clusters comprised strains that were clearly different by IS6110 RFLP genotyping. Cluster M2 from their study compiles 24 strains and cluster M11 10 strains, from which each strain represents a unique RFLP profile. In our study, clusters SK2 and SK1 correspond to clusters M2 and M11, respectively. Unfortunately, the frequencies of SK1 and SK2 in the Ural region cannot be compared to the MIRU clusters in North-West Russia, because of the different criteria used to form the collections (convenience sample and selected unique Beijing RFLP profiles, respectively). It is interesting to note that four of 50 isolates from the W-Beijing family k64, k166 (both from SK1C), k26 (SK3A) and k36 (SK3B) have no IS6110 copy in the genome NTF locus. The absence of the insertion in NTF could be a sign of evolutionary heterogeneity within the family. The results of the phylogenetic analysis confirm this supposition (Figure 2): these isolates are phylogenetically divergent from other clustered isolates of the W-Beijing family. According to our data, and taking into account the earlier published suppositions that the number of IS6110 copies in the genome of M. tuberculosis does not decrease, it could be assumed that the isolates of the W-Beijing type that circulate in the Ural region are related to old or ancient (NTF without IS6110 insertion) as well as young isolates (IS6110 in NTF) with significant domination by the latter group. This is in good agreement with recent data published for North-West Russia. 8,26 The IS6110 insertion in the NTF locus in some isolates (k71, k11 and k91) that belong to the evolutionarily distinct groups Ural and Others (Figure 1) could be identified by independent transposition of the IS6110 element, and its exact location in NTF locus needs to be confirmed by sequencing. Drug resistance analysis of the Ural regional collection of M. tuberculosis strains showed that resistance to any anti-tuberculosis drugs among newly diagnosed patients (44.1%) is higher than the average resistance reported for the other regions of the Russian Federation (15 35%). 27 W-Beijing isolates resistant to at least one drug were twice as frequent as in other groups (Table 2). Moreover, 19/23 (82.6%) MDR strains were also members of the W-Beijing family. Our data therefore confirm the previously found

6 Genetic analysis of M. tuberculosis by MIRU-VNTR 751 association of this genotype with multidrug resistance. 28,29 Taking into account that resistance to one drug does not dramatically worsen the prognosis for TB treatment outcome, the spread of M. tuberculosis isolates of the Ural and Others groups seems to be less rapid in the Ural region. In conclusion, MIRU-VNTR genotyping of M. tuberculosis isolates from 92 patients with PTB revealed several major groups of strains circulating in the Ural region, with MDR-TB associated W-Beijing strains predominating. MIRU-VNTR typing, as well as IS6110 RFLP genotyping and spoligotyping in other studies, is a reliable and powerful tool for molecular epidemiological studies of M. tuberculosis. The establishment of national databases of MIRU- VNTR and RFLP profiles of M. tuberculosis isolates and their comparison with international databases will allow us to create a complete picture of the transmission of M. tuberculosis strains from different groups and families throughout the country, to reveal the regional and local characteristics of causative agent circulation and to answer questions concerning the sources of infection, global TB dissemination and probable measures of control for TB. Acknowledgements We are grateful to P Bifani, O Narvskaya and I Mokrousov for critical reading and W Eisner for assistance in preparing the manuscript. References 1 Sverdlovsk Regional Center for Sanitary and Epidemiologic Control. Data of TB morbidity, Ekaterinburg, Sverdlovsk: Sverdlovsk Regional Center for Sanitary and Epidemiologic Control, jan_dec_2003.html Accessed November Barnes P F, Cave M D. Molecular epidemiology of tuberculosis. N Engl J Med 2003; 349: Kremer K, van Soolingen D, Frothingham R, et al. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J Clin Microbiol 1999; 37: Supply P, Mazars E, Lesjean S, et al. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 2000; 36: Supply P, Lesjean S, Savine E, et al. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 2001; 39: Frothingham R, Meeker-O Connell W A. Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 1998; 144(Pt 5): Medvedeva T V, Ogarkov O B, Nekipelov O M, et al. Genotyping of strains of M. tuberculosis circulating in Eastern Siberia (Russia). Moscow, Russian Federation: Russian Society of Phthisiatrician, Mokrousov I, Narvskaya O, Limeschenko E, Vyazovaya A, Otten T, Vyshnevskiy B. Analysis of the allelic diversity of the Mycobacterial Interspersed Repetitive Units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations. J Clinical Microbiol 2004; 42: Viljanen M K, Vyshnevskiy B I, Otten T F, et al. Survey of drugresistant tuberculosis in northwestern Russia from 1984 through Eur J Clin Microbiol Infect Dis 1998; 17: Yeboah-Manu D, Yates M D, Wilson S M. Application of a simple multiplex PCR to aid in routine work of the mycobacterium reference laboratory. J Clin Microbiol 2001; 39: Mazars E, Lesjean S, Banuls A L, et al. High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci USA 2001; 98: Skuce R A, McCorry T P, McCarroll J F, et al. Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology 2002; 148(Pt 2): Roring S, Scott A, Brittain D, et al. Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping. J Clin Microbiol 2002; 40: Plikaytis B B, Marden J L, Crawford J T, et al. Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis. J Clin Microbiol 1994; 32: Hunter P R, Gaston M A. Numerical index of discriminatory ability of typing system: an application of Simpson s index of diversity. J Clin Microbiol 1988; 26: Swofford D L. PAUP*. Phylogenetic analysis using parsimony (*and other methods). 4th ed. Sunderland, MA: Sinauer Associates, Supply P, Warren R M, Banuls A L, et al. Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area. Mol Microbiol 2003; 47: Institut Pasteur de Lille. MIRU-VNTR home page. Lille, France: INSERM U447, Institut de Biologie de Lille, Accessed November World Health Organization. Russian Federation, Global TB Control Report. Geneva, Switzerland: WHO, int/tb/publications/global_report/2004/en/russianfederation.pdf 20 Espinal M A. The global situation of MDR-TB. Tuberculosis (Edinb) 2003; 83: Wise J. WHO identifies 16 countries struggling to control tuberculosis. BMJ 1998; 316: Narvskaia O V, Mokrousov I V, Limeshchenko E V, et al. Characterization of Mycobacterium tuberculosis strains prevalent in North-West of Russia by spoligotyping. Probl Tuberk 2002; 4: Drobniewski F, Balabanova Y, Ruddy M, et al. Rifampin- and multidrug-resistant tuberculosis in Russian civilians and prison inmates: dominance of the Beijing strain family. Emerg Infect Dis 2002; 8: Kurepina N, Shashkina E, Ramasvamy S, et al. A primary multidrug resistant W-Beijing Mycobacterium tuberculosis cluster predominates in a West Siberian prison population (Russia). Cascais, Portugal: Second Meeting of the Concerted Action Project May 2002: p Rad M E, Bifani P, Martin C, et al. Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the W-Beijing family. Emerg Infect Dis 2003; 9: Mokrousov I, Narvskaya O, Otten T, et al. Phylogenetic reconstruction within Mycobacterium tuberculosis Beijing genotype in northwestern Russia. Res Microbiol 2002; 153: Sevostyanova E V, Shul gina M V, Puzanov V A, Martynova L P, Golyshevskaya V I, Erokhin V V. Drug resistance analysis of Mycobacterium tuberculosis in experimental regions introducing WHO tuberculosis program. Probl Tuberk 2002; 12: Bifani P J, Mathema B, Kurepina N E, Kreiswirth B N. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. 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7 752 The International Journal of Tuberculosis and Lung Disease RÉSUMÉ CONTEXTE : La région de l Oural (Russie) est une des zones où l incidence de la tuberculose (TB) est élevée. Dans ces circonstances, les études d épidémiologie moléculaire aptes à dépister les voies de dissémination de Mycobacterium tuberculosis ont une signification particulière. OBJECTIF : Caractérisation de la population de souches de M. tuberculosis circulant dans la région de l Oural, détection des génotypes prédominants et évaluation de leur relation phylogénique et de leur signification épidémiologique. SCHÉMA : On a soumis à un génotypage par la méthode MIRU-VNTR 92 échantillons cliniques de M. tuberculosis provenant de la région de l Oural. RÉSULTATS : On a pu identifier deux groupes principaux phylogénétiquement distincts : la famille W-Beijing (54,3%) et une grappe antérieurement non signalée dénommée le groupe Oural (15,2%). On a pu identifier 47 profils MIRU différents, y compris 38 isolés (41,3%) et 9 groupés en grappes comportant chacune de 2 à 28 isolats et correspondant à 54 cas. La diversité génétique au sein des grappes a été démontrée par un sous-typage additionnel d isolats de M. tuberculosis dans neuf loci complémentaires QUB-VNTR. CONCLUSION : Il a été démontré que les isolats de la famille W-Beijing sont associés à une multirésistance à l égard des médicaments antituberculeux. Il est possible que les souches de cette famille jouent un rôle significatif dans la dispersion de la TB à germes multirésistants dans la région de l Oural. RESUMEN MARCO DE REFERENCIA : En la Federación de Rusia, la región Ural es una de las zonas con mayor incidencia de tuberculosis (TB). En estas circunstancias, adquieren particular importancia los estudios de epidemiología molecular que permitan detectar las rutas de diseminación de la TB. OBJETIVO : Caracterizar la población de cepas de Mycobacterium tuberculosis que circulan en la región Ural, detectar los genotipos predominantes y evaluar su relación filogenética y su significación epidemiológica. MÉTODO : Se analizó el genotipo de 92 aislados clínicos de M. tuberculosis provenientes de la región Ural, con base en el método MIRU-VNTR. RESULTADOS : Se identificaron dos grupos principales independientes filogenéticamente : la familia W-Beijing (54,3 %) y un conglomerado no descrito previamente denominado el grupo Ural (15,2 %). Se identificaron 47 perfiles diferentes de MIRU, entre ellos 38 aislados micobacterianos únicos (41,3 %) y 54 aislados agrupados en 9 conglomerados (de 2 a 28 aislados en cada conglomerado). Se puso en evidencia una diversidad genética en cada conglomerado mediante subtipificación de los aislados de M. tuberculosis en nueve locus QUB-VNTR adicionales. CONCLUSIÓN : Se demostró que los aislados micobacterianos de la familia W-Beijing se asocian con farmacorresistencia múltiple a los medicamentos antituberculosos. Es posible que las cepas de esta familia jueguen un papel significativo en la diseminación de la TB con farmacorresistencia múltiple a través de la región Ural.