Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation

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1 Eur J Clin Microbiol Infect Dis (2011) 30: DOI /s z ARTICLE Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation M. V. Afanas ev & L. N. Ikryannikova & E. N. Il ina & A. V. Kuz min & E. E. Larionova & T. G. Smirnova & L. N. Chernousova & V. M. Govorun Received: 14 May 2010 / Accepted: 9 September 2010 / Published online: 13 October 2010 # Springer-Verlag 2010 Abstract The present study investigates epidemiological diversity and multidrug resistance spreading among Mycobacterium tuberculosis strains circulating in Moscow, Russian Federation. Among 115 M. tuberculosis strains selected randomly from the sputum of epidemiologically unrelated tuberculosis (TB) patients, multidrug-resistant (MDR) strains predominated. Mutations in the RRDR of the rpob gene were detected in 64 (83.1%) of 77 rifampicin (RIF)-resistant strains. The Ser531 Leu substitution was prevalent among them (76.5%). Aberrations in the Ser315 codon of katg and/or in the inha promoter region were found in 79 (84.0%) of 94 isoniazid (INH)- resistant strains. Strains belonging to the Beijing family prevailed. Seventy-one different patterns were identified using the 24-VNTR loci typing scheme. Three main 24- loci VNTR clusters included 34 strains which belonged to the Beijing family. The spoligotyping and 24-loci VNTR typing combination demonstrated maximal discriminatory power. Among the Beijing strains, the MDR phenotype Electronic supplementary material The online version of this article (doi: /s z) contains supplementary material, which is available to authorized users. M. V. Afanas ev (*) : L. N. Ikryannikova : E. N. Il ina : V. M. Govorun Research Institute of Physical-Chemical Medicine of the Ministry of Public Health of the Russian Federation, Malaya Pirogovskaya st., 1a, Moscow, Russia afanasev_max@mail.ru A. V. Kuz min : E. E. Larionova : T. G. Smirnova : L. N. Chernousova Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, Moscow, Russia was revealed more frequently than among the others. High genetic heterogeneity of the studied population was shown by the assessment of VNTR loci variability in the analyzed group and in the strains from other parts of Russia. Comparison of the 24-VNTR locus typing and spoligotyping data with revealed resistance-associated mutation allows us to make a suggestion that the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy occurred in the studied population simultaneously. Introduction Moscow is the capital of the Russian Federation, with more than ten million inhabitants. It is the largest city in the country and the major industrial and transporting center with rather intensive migration flows (including people from Former Soviet Union [FSU] countries). In Moscow, tuberculosis (TB) morbidity amounted to about 50 cases per 100,000 people in High TB morbidity rate and increasing numbers of multidrugresistant (MDR) (defined as resistance at least to isoniazid and rifampicin) strains [1] attract attention to the effective preventive, prophylactic, and surveillance measures aimed to prevent and avoid M. tuberculosis transmission and spreading of the MDR strains in the community. The main goal of this study was to characterize the population of M. tuberculosis strains circulated among Moscow residents by molecular typing-based methods and to detect the phylogenetic relationship between strains and epidemiological significance of particular genotypes. During the investigation, we employed for typing the 24- loci set of VNTR loci as previously described [2] in

2 182 Eur J Clin Microbiol Infect Dis (2011) 30: combination with spoligotyping [3] for more comprehensive analysis of the mycobacterial population. Minisequencing reaction followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) of the reaction products was used to detect nucleotide substitutions associated with rifampicin (RIF) and isoniazid (INH) resistance, as we have previously described [4, 5]. The combination of genotyping and drug resistance marker detection enables us to receive a more complete picture of the characteristics of the TB strains circulated in the Moscow region. Materials and methods Sample collection This study included 115 M. tuberculosis strains. These strains were selected randomly from M. tuberculosis strains revealed from TB patients during the years in the Microbiology Department, Central Tuberculosis Research Institute of the Russian Academy of Medical Sciences (RAMS, Moscow, Russia). Strains were isolated from the sputum of HIV-negative, epidemiologically unrelated lung TB patients who were Moscow residents (constant registration in Moscow and absence of departures outside Moscow and the Moscow region during the last five years). Demographic and social data, and past and present medical TB history were collected for each patient. M. tuberculosis strains identification and drug susceptibility testing Strains isolation and identification were performed according to the World Health Organization (WHO) recommendations [6]. RIF and INH susceptibility tests were carried out by the absolute concentration method on Löwenstein Jensen medium containing 40 mg/l of RIF or 1 mg/l of INH, respectively. All of the drugs were manufactured by Sigma, USA. The results of the microbiological tests were registered 21 days after the inoculation. Isolates were considered to be resistant when more than 20 colonies grew on the drug-containing medium. Laboratory strain M. tuberculosis H37Rv was used as a control for all microbiological and genetic procedures. DNA isolation The DNA of M. tuberculosis was extracted by the Polytub kit (Lytech Ltd., Russia), based on the method of Boom et al. [7], in accordance with the manufacturer s instructions. Genetic drug resistance analysis Genetic detection of RIF and INH resistance was performed using non-commercial systems based on the minisequencing reaction followed by MALDI-ToF MS analysis. This system included 18 probes for the detection of the most frequent mutations in the rpob and katg genes, and the inha promoter region [4, 5]. The set of probes for the rpob gene comprised 13 oligonucleotides for the detection of both wild-type genotypes (i.e., no mutations) and all possible nucleotide substitutions in the Leu533, Ser531, His526, Ser522, Asp516, Phe514, Gln513, and Leu511 codons. The sets of probes for the katg gene and the inha promoter region comprised five oligonucleotides, indicating either wild-type genotype or mutations in codon Ser315 katg or in the -8 and -15 inha promoter positions, respectively. Strains were considered as RIF-resistant when mutations in the rpob gene were detected. Strains with mutation in the katg and/or inha promoter region were reckoned as INH-resistant. Strains with mutations in both the rpob and katg (and/or inha promoter) loci were regarded as MDR. For validation of the minisequencing reaction followed by MALDI-ToF MS analysis results, direct sequencing of the rpob, katg, and inha promoter loci was carried out for some randomly selected strains. Sequencing reactions were performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, USA; Hitachi, Japan) according to the manufacturer s instructions. Genotyping methods The number of tandem repeats was determined in 24-VNTR genetic loci: 12 original MIRU-VNTR loci, three loci of exact tandem repeats (ETRs): ETR-A, -B, and -C, six Mtub loci: Mtub04, 21, 29, 30, 34, and 39, and three Queen suniversity Belfast (QUBs) loci: QUB-11b, -26, and 4156c. Polymerase chain reaction (PCR) amplification was performed as described for correspondent loci in the previous studies [8 12]. The PCR products were evaluated by electrophoresis in 1.5% agarose gel and the number of repeats was calculated from the fragments sizes by comparison with GeneRuler 100-bp DNA Ladder (Fermentas, Lithuania) used as a fragment size standard, and the VNTR Calculation module in Quantity One v software (BioRad, USA). The accuracy of the calculation was checked through simultaneous analysis of the M. tuberculosis H37Rv strain. Spoligotyping was performed as described previously [3], using biotinylated primers for DR-region amplification and nylon membranes with immobilized spacers probes from the spoligotyping kit from Isogen Lifescience (cat. no. IM9701).

3 Eur J Clin Microbiol Infect Dis (2011) 30: Spoligotypes in binary format were entered in an Excel spreadsheet and compared with the spoligotyping database SpolDB4 [13]. The online database SITVIT (Institute Pasteur de Guadeloupe; fr:8081/sitvitdemo/outilsconsultation.jsp, last revision October, 2008) was used to determine spoligotype international type (SIT) numbers. Statistical analysis The analysis of the genotyping data was performed using Statistica 6.0 software (StatSoft Ink., USA). The chi-square test or two-tailed Fisher s exact test were used for bivariate contingency tables analysis; p-values less than 0.05 were considered to be statistically significant. The allelic diversity (h) at a given VNTR locus was calculated as 1 P x 2 i ½n= ðn 1ÞŠ; where x i is the frequency of allele i in the locus and n is the number of strains with the i allelic variant. The Hunter Gaston discriminatory index (HGDI) was used to calculate the discriminative power of each genotyping method [14]. The clustering rate (%) was defined by the formula ðt c N c =T a Þ100%; where T c is the number of clustering strains, N c is the number of clusters, and T a is the total strains number. The public online resource MIRU- VNTRplus ( faces, last revision October, 2009) and online instruments [15] were used for phylogenetic trees construction (by the unweighted pair group method using arithmetic averages [UPGMA] algorithm) and minimum spanning tree generation. Results Population structure In total, we have analyzed 115 lung TB patients who had been treated in the clinic of the Central Tuberculosis Research Institute, Russian Academy of Medical Sciences (Moscow, Russia) during the years All patients were HIV-negative. Of these, male (76.5%), newly diagnosed (71.3%), non-destructed clinical forms (62.6%), and younger than 50 years of age (75.6%) patients predominated. There was no significant difference between the demographic and clinical parameters among patients infected with Beijing or non-beijing strains (Table 1). Susceptibility testing Among 115 M. tuberculosis strains included in the study, 76 (66.0%) were identified as MDR (i.e., resistant to both RIF and INH), 20 (17.4%) were susceptible to RIF and INH, and 1 (0.9%) and 18 (15.7%) isolates were resistant, respectively, either to RIF or INH. Detection of mutations in the rpob and katg genes and the inha promoter region Mutations in the RRDR of the rpob gene were detected in 64 (83.1%) of 77 RIF-resistant M. tuberculosis isolates. Ten different types of mutations were identified in the Leu533, Ser531, His526, and Asp516 codons and also one variant insertion between codons 514 and 515. No substitutions in Ser522, Gln513, and Leu511 were detected for the investigated M. tuberculosis strains. Mutation TCG TTG (Ser Leu) in codon 531 was the most prevalent; it was detected in 47 (73.4%) of 64 RIF-resistant strains. Three different types of point nucleotide substitutions were found in the His526 codon of five (7.8%) strains. Seven (10.9%) strains revealed three variants of mismatches in Asp516. Two (3.1%) strains carried mutations in Leu533. Insertion Phe514a was found for only one M. tuberculosis strain. Two strains carried point mutations in two separated codons, Ser 531 and His526. Substitutions in the Ser315 codon of katg and/or the inha promoter region were found in 79 (84.0%) of 94 INHresistant M. tuberculosis strains. Most of these strains, 56 (59.6%), had mutation Ser315 Thr (AGC ACC). Twenty-two (23.4%) strains had point nucleotide mutations in the 15th and 8th positions of the inha promoter region. Twenty of them also contained substitutions in Ser315 of katg. VNTR genotyping of M. tuberculosis clinical strains Twenty-four-VNTR loci, constituent to a recently proposed scheme [2], were used for the typing of all 115 of the studied clinical M. tuberculosis strains. Seventy-one different VNTR profiles were detected, 59 (83.0%) of them were unique (i.e., observed for only one strain). Fifty-six strains were grouped into 12 clusters, each including from 2 to 13 strains. Three main clusters contained 13 (11.3%), 12 (10.4%), and 9 (7.8%) M. tuberculosis strains that had , , and VNTR profiles, respectively (hereinafter, MIRU-VNTR loci are listed according to their position in kbp on the H37Rv genome, from 154 kbp for MIRU 02 to 4,338 kbp for MIRU 39). The allelic diversity of different MIRU-VNTR loci varied widely from 0 to Seven loci (MIRU 04, MIRU 20, MIRU 23, MIRU 27, ETRB, Mtub 29, and Mtub 34) were almost monomorphic (0 < h < 0.1). Locus MIRU 26 was absolutely invariable among all of the studied strains. Six loci, MIRU 26, MIRU

4 184 Eur J Clin Microbiol Infect Dis (2011) 30: Table 1 Demographic and social characteristics of tuberculosis (TB) patients from whom the studied Mycobacterium tuberculosis strains were isolated. Two groups infected with Beijing and non-beijing strains are considered separately a Statistical significance of the difference between Beijing and non-beijing strains b According to verbal information provided by the patients Category No. of patients (%) Total: 115 (100) Beijing: 76 Non-Beijing: 39 p-value a Sex Male 88 (76.5) Female 27 (23.4) Age, mean (SD), [range], years 39.4 (14.3), [15 70] <20 7 (6.0) (27.8) (23.5) (18.3) (15.7) >60 10 (8.7) Form of TB Disseminated 9 (7.8) Infiltrative 61 (53.0) Infiltrative with destruction 23 (20.0) Cavernous (fibrocavernous) 17 (14.8) Caseous pneumonia 3 (2.6) Tuberculoma 2 (1.7) TB treatment Newly diagnosed 82 (71.3) Previously treatment 33 (28.7) 26 7 Former prisoner 7 (6.0) Contact with TB patients (family, professional, prison) b 14 (12.2) , ETRA, QUB-11b, and QUB-26, demonstrated high variability (h>0.5) (Table 2). The discriminatory power of the traditional 12-loci MIRU typing was less than 0.9. For 15-loci and 24-loci sets, the HGDI values were higher, with differences between them being insignificant (0.965 and 0.968, respectively). Spoligotyping Spoligotyping showed 26 different genotypes, clustered into 15 genetic families, with 97 (84.3%) strains in seven clusters (from 2 to 73 strains), and 18 strains with unique genotypes. Strains belonging to the Beijing family formed the largest cluster of SIT1 (73/115; 63.4%). Three strains belonged to a Beijing-like family. The next two largest families (LAM and T) rarely occurred (10.4% and 8.7%, respectively). Six strains belonged to unknown spoligotypes not present in the SpolDB4 database (Table 3). As expected, the HGDI value for spoligotyping was extremely low (0.592), especially among Beijing family strains (0.077). All information about the studied strains is presented in Supplementary Table 1. Discussion M. tuberculosis infection still remains an acute health problem in the Russian Federation. Although TB morbidity has decreased slightly in the last several years, the situation remains alarming due to a major increase in the incidence and prevalence of MDR-TB [16]. Clearly, prompt measures for TB identification and differentiation including molecular genetic typing techniques are needed to prevent TB from becoming widespread in the human population. Many researchers used the spoligotyping technique, conventional molecular typing, e.g., IS6110 restriction fragment polymorphism (RFLP) analysis, and only recently have introduced 12-loci MIRU typing to characterize genetic diversity and prevalent genotypes in M. tuberculosis populations circulating in different regions of the Russian Federation [17 20]. Genetic analysis of drug resistance combined with spoligotyping, traditional 12-loci MIRU typing, and the newly proposed 15- and 24-loci VNTR typing formats, which demonstrated a higher discriminatory power [2], were used in the present work (i) to reveal molecular genetic features of M. tuberculosis strains circulating in Moscow and to investigate the power of various typing

5 Eur J Clin Microbiol Infect Dis (2011) 30: Table 2 Variability of 24-VNTR loci among the studied clinical M. tuberculosis strains (n=115) MIRU-VNTR convention Alias Involved in different schemes Size range observed (copy number) 12-loci VNTR 15-loci VNTR No. of alleles observed Index of allele polymorphism; h 154 MIRU Mtub ETRC MIRU MIRU MIRU MIRU Mtub MIRU b QUB-11b ETRA Mtub Mtub ETRB MIRU MIRU MIRU MIRU Mtub MIRU Mtub QUB QUB-4156c MIRU methods for the differentiation of the M. tuberculosis population in Moscow, and (ii) to estimate the role of strains with different genotypes in TB (especially, MDR) spread. Molecular genetic description of M. tuberculosis strains circulating in Moscow and evaluation of the discriminatory power for various typing methods As it has been previously observed in many studies, the Beijing family was prevalent among strains circulating in different regions of Russia [17 20]. This family is also widespread in many countries around the world [21] and demonstrates some significant clinical and epidemiological properties, such as high transmissibility, increased virulence, and MDR phenotype association [22 24]. According to the previous observations, in the central part of Russia, including districts neighboring Moscow (Tula and Serpukhov), besides Beijing strains, strains of the LAM [19] and Haarlem [25] families are also prevalent. In the studied microbial population, the Haarlem family numbered only four (3.4%) strains, whereas the T family constituted the third largest group (8.7%) and was inferior only to the Beijing and LAM families. By comparison of the presented data with other studies, it was found out that the highest prevalence of the Beijing family in Russia occurred in the Samara region (66.6%) [26], Eastern Siberia (Irkutsk) (63.2%) [20], and Western Siberia (Novosibirsk) (60.7%) [27]. In the north-western region (St. Petersburg) and Ural (Yekaterinburg), the Beijing strain frequency was near 54% and 55.9%, respectively [17, 28]. The occurrence of this family was lower in the westernmost part of the Russian Federation (Kaliningrad), being 45% [18], and the Central area of Russia (Tula and Serpukhov), being 42.4% [19]. In FSU countries, the frequency of the Beijing family varied from 39.6% (Ukraine) [29] to 42.0% and 53.5% (Estonia and Latvia, respectively) [22, 30]. Thus, the rate of Beijing strains among Moscow residents was higher than in most of other the Russian areas and FSU countries. As it is well known, spoligotyping could not effectively distinguish mycobacterial populations in the regions with predominant or endemic strains which demonstrate considerable genetic homogeneity [31].

6 186 Eur J Clin Microbiol Infect Dis (2011) 30: Table 3 Spoligotypes observed in the studied clinical M. tuberculosis strains (n=115) Family SIT number a No. of strains (%) Beijing 1 73 (63.4) (0.85) (1.7) LAM (0.85) LAM (6.9) (0.85) 252 2(1.7) LAM 3 and S/convergent 4 1 (0.85) T (5.2) (0.85) T (0.85) T2 T (0.85) T (0.85) T1_RUS (0.85) T 5_RUS (2.6) H (0.85) H (0.85) H (1.7) U (0.85) U (likely H 3) (0.85) a SIT spoligotype international type Thereby, to increase the discriminatory power of genotyping methods and for a better understanding of the molecular diversity of the studied population, the VNTR typing procedure has been applied as well. We used different schemes of VNTR analysis: traditional 12- loci MIRU typing and 15- and 24-loci VNTR typing schemes. Twelve-loci MIRU analysis revealed 12 clusters (from 2 to 27 strains), with eight groups among them being formed by Beijing strains. The two largest clusters contained 27 and 25 members, respectively, and had MIRU-VNTR profiles and (M2 and M11 genotypes, according to the nomenclature suggested by Mokrousov et al. [28]. All of these strains also belonged to the Beijing family. In the Samara region, the central part of Russia, significant prevalence of the profile (M2) among prisoners and profile (M11) among civilians was described by Drobniewski et al. [26]. In our setting, no significant association was observed between prison or civil status and MIRU genotype. Moreover, among seven strains isolated from former prisoner TB patients included in our study, six had genotype (M11), which is more typical for civilians. The homogeneity of the Beijing population observed in Russia is caused by recent dissemination of the circulating Beijing clone [32], which also suggests low VNTR loci variability detected among Russian Beijing strains [33]. Thus, for example, in the Ural region for the 12-MIRU- VNTR typing system applied to Beijing strains, the value of HGDI was 0.64 and the mean of the allele polymorphism index for 12-MIRU loci was [17]. In the Kaliningrad region, these values were and [18], and in the north-western regions, they were 0.65 and , respectively [28] (Table 4). In their recent work, Mokrousov et al. [33] observed greater values of HGDI and the mean of the allele polymorphism index, being and , respectively. In the present work, we have revealed a great heterogeneity among Beijing strains the HGDI value was 0.8 and the mean allele polymorphism index was (for the 12- MIRU loci set). We suppose that the high heterogeneity of the Beijing population formed in the largest Russian cities Moscow and St. Petersburg at present time is associated with an increasing level of population migration from other parts of the Russian Federation and FSU countries. Fifteen- and 24-VNTR loci schemes demonstrated higher discriminatory power (0.965 and 0.968, respectively, for the total set, and and for Beijing strains). The best discriminatory power was demonstrated by a combination of 24-loci VNTR analysis and spoligotyping (the HGDI value was for all strains and was for Beijing strains only) (Table 5) (Fig. 1). Based on 24-loci VNTR, all Beijing strains under investigation were divided into three clonal complexes (CCs), which consisted of strains that differed from each other by a single locus change. The 24-loci VNTR-based minimum spanning tree of the VNTR types within the Beijing family is presented in the Fig. 2. Three additional subgroups were separated in the CC1 cluster. These subgroups contained subclones formed by strains with identical VNTR genotypes, from 9 to 13 strains per group. The complexity of the population structure, the presence of three CCs, and three subgroups within CC1 also gives evidence of quite a high genetic heterogeneity. We have also examined the distribution of mutations in the rpob gene, the katg gene, and the inha promoter region in the studied population. Expectedly, substitutions in Ser531 rpob were prevalent among RIF-resistant strains, as known to be typical for many world regions [34 36], and were previously observed in our own investigation [5]. It is necessary to mention that, in the present study, among TB strains circulated in Moscow, Asp516 rpob mutations were observed more frequently than substitu-

7 Eur J Clin Microbiol Infect Dis (2011) 30: Table 4 Twenty-four-VNTR loci diversity in Beijing family strains from the Russian Federation MIRU-VNTR convention Alias Index of allele polymorphism; h This study Kaliningrad [18] St. Petersburg [33] West Siberia [27] Ural a [17] 154 MIRU Mtub ETRC MIRU MIRU MIRU MIRU Mtub MIRU b QUB-11b ETRA Mtub Mtub ETRB MIRU MIRU MIRU MIRU Mtub MIRU Mtub QUB QUB-4156c MIRU a For Beijing strains represented by clusters only (five non-clustered Beijing strains are excluded) Table 5 Comparative analysis of genotyping methods applied to study clinical M. tuberculosis strains (n=115) circulating in the Moscow region, Russia Typing method, strain groups No. of genotypes in total No. of unique genotypes a No. of clusters No. of strains in clusters HGDI value Spoligotyping All strains Beijing strains only Non-Beijing strains VNTR analysis, all strains 24-VNTR MIRU-VNTR Spoligo + 24-VNTR All strains Beijing strains only Spoligo + 12-MIRU-VNTR All strains Beijing strains only a Observed for one strain only

8 188 Eur J Clin Microbiol Infect Dis (2011) 30: VNTR pattern spoligopattern

9 Eur J Clin Microbiol Infect Dis (2011) 30: Fig. 1 Distribution of the clinical Mycobacterium tuberculosis strains (n=115) based on unweighted pair group method using arithmetic averages (UPGMA) clusterization of their individual 24-loci VNTR patterns. MIRU-VNTR loci are listed according to their position in H37Rv genomic DNA, from 154 kbp for MIRU 02 to 4338 kbp for MIRU 39 (see also Tables 2 and 4) tions in His526 rpob, and the 516 codon was as variable as that of 526. We think that this can be explained by the fact that the broader Central area of the Russian Federation and not only the Moscow region was involved in the previous work [5], where the typical frequency of distribution of drug resistance-associated alterations in the rpob gene (Ser531>His526>Asp516) had been observed. Ser531 rpob mutations were revealed more frequently among Beijing strains (48/57; 84.2%) than among non- Beijing strains (1/7; 14.3%) (Chi-square=16.99, p= ). Likewise, His526 rpob substitutions were associated with the LAM family (4/7; 57.1%) (Chi-square=26.56, p=0.0003), and no association between alterations of Asp516 of the rpob gene and spoligotype was found. For the INH-resistant Beijing strains isolated, Ser315 katg mutations were more typical (54/67; 80.6%), while strains from other families had simultaneous mutations in Ser315 katg and the inha promoter region (10/12; 83.3%). The role of strains with different genotypes in TB (especially MDR) spread Based on 24-loci VNTR analysis and spoligotyping, 52 strains were grouped into 11 clusters (Fig. 1). Ten of them included strains belonging to the Beijing family only. The clustering rate was 49.8% among Beijing strains and only 2.5% among non-beijing strains. In our study, MDR phenotype was revealed more frequently among Beijing strains (59/76; 77.6%) than among other families (17/39; 43.6%) (Chi-square=13.3; p=0.0003). Consequently, drug resistance-associated mutations in clustered groups were observed more Fig. 2 The 24-loci VNTR-based minimum spanning tree within the clinical M. tuberculosis strains belonging to the Beijing family (n= 76). CC = clonal complexes. Numerals printed over the lines indicate the number of locus changes. Circle A includes strains Ly-51, 56, 68, 70, 73, 74, 80, 179, 189, 192, 392, 412; B includes strains Ly-72, 77, 81, 84, 85, 186, 188, 191, 199, 205, 350, 367, 380; C includes strains Ly-75, 83, 86, 190, 200, 366, 388, 395; D includes strains Ly-193, 349, 385; E includes strains Ly-79, 381, 397; F includes strains Ly-54, 379; G includes strains Ly-185, 187, 194; H includes strains Ly-390, 393, 410; I includes strains Ly-52, 57

10 190 Eur J Clin Microbiol Infect Dis (2011) 30: frequently than in non-clustered groups, and the clusterization level may be considered as an indirect sign of TB transmission in the population. This was the reason to make an attempt to estimate the role of Beijing strains in TB spread among different groups of patients. First of all, we reviewed strains (n=82/115; 71.3%) isolated from primarily revealed patients who had not been treated previously. The level of clusterization for Beijing strains among this group was quite high (40%), but for non- Beijing stains, it was only 3.1%. Grouping strains into clusters was observed more frequently among Beijing strains than among non-beijing ones (Chi-square=13.83, p=0.0002). Also in the selected group, clustered MDR Beijing strains were revealed significantly more frequently than non-clustered strains (p=0.0076). The high level of clusterization for MDR Beijing strains isolated from previously non-treated patients can be considered as evidence of the active transmission of such strains in the human population. On the other hand, one needs to keep in mind that clusterization can also reflect the persistence of well-conserved endemic strains only. The small number of samples, short observation period, and absence of comprehensive epidemiological data are serious limiting factors of this investigation that have caused bias in the estimation of results [37]. For a better understanding of the drug resistance spreading mechanism, we have analyzed patterns of mutations associated with resistance in strains with particular genotypes which belonged to different CCs or CC1 subgroups. The identical mutations in the rpob and katg genes (except one strain only) were detected in strains (n=11) forming the same 24-loci VNTR subgroup 2, generated in CC1 (see Fig. 2). We suggested that such phenomena demonstrated that these substitutions had not been acquired due to chemotherapy, but warned about the active transmission of MDR strains belonging to this cluster. On the other hand, the observation of different mutations in the rpob and katg genes, and the inha promoter region in subgroups 1 and 3 may testify to the independent appearance of these alterations during chemotherapy and/or different capacity of the resistance acquisition within certain genotypes. Most likely, both processes occurred in the studied population. In conclusion, we need to state that the present work is the first molecular snapshot of the clinical M. tuberculosis strains circulating in Moscow, the capital of the Russian Federation. We have demonstrated the heterogeneity of the Moscow M. tuberculosis population in comparison with other Russian regions. Our results highlight some features of MDR M. tuberculosis spreading in the region. The analysis of the association of certain mutations with particular genotypes testifies to the existence of two parallel processes: the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy. This study should be continued and extended to other Russian cities and regions for a better understanding of the M. tuberculosis diversity and control of drug-resistant TB spread in the human population. Acknowledgments We are grateful to V.A. Karpov for the oligonucleotide primers synthesis. References 1. World Health Organization (WHO) (2008) Anti-tuberculosis drug resistance in the world. Fourth global report. WHO, Geneva 2. 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