Molecular snapshot of drug-resistant and drug-susceptible Mycobacterium tuberculosis strains circulating in Bulgaria

Infection, Genetics and Evolution 8 (2008) 657–663

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Molecular snapshot of drug-resistant and drug-susceptible Mycobacterium tuberculosis strains circulating in Bulgaria Violeta Valcheva a, Igor Mokrousov b, Olga Narvskaya b, Nalin Rastogi c,*, Nadya Markova a,** a

Department of Pathogenic Bacteria, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Laboratory of Molecular Microbiology, St. Petersburg Pasteur Institute, 197101 St. Petersburg, Russia c Unite´ de la Tuberculose et des Mycobacte´ries, Institut Pasteur de Guadeloupe, Abymes 97183, Guadeloupe b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 June 2008 Received in revised form 26 June 2008 Accepted 26 June 2008 Available online 9 July 2008

We report results of the first study on the molecular basis of drug resistance in Mycobacterium tuberculosis strains currently circulating in Bulgaria. The study panel consisted of 133 (including 37 drug-resistant) isolates recovered from newly diagnosed, adult pulmonary TB patients from different regions of Bulgaria in 2005–2006. Three types of the rpoB mutations were found in 20 of 27 RIF-resistant isolates; rpoB S531L was the most frequent. Eleven (48%) of 23 INH-resistant isolates had katG S315T mutation. inhA 15C > T mutation was detected in one INH-resistant isolate (that also had katG315 mutation) and three INHsusceptible isolates. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. Comparison with spoligotyping and 24-VNTR locus typing data suggested that emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any specific spoligotype or MIRU-VNTR genotype. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Drug resistance Mutation Spoligotyping VNTR

1. Introduction Tuberculosis (TB) infects a significant proportion of the world population and constitutes a major public health problem, particularly, in the developing regions. A reemergence of TB accompanied by an increasing number of drug-resistant and multidrug-resistant (i.e. resistant to at least RIF and INH) Mycobacterium tuberculosis strains has been noted since the mid-1980s. Although a number of new TB cases in Bulgaria is showing a steady decline since 2001 (48.6/100,000), the TB notification rate is still sufficiently high (41/100,000 in 2006) (WHO, 2008b). The rate of the MDR-TB among newly diagnosed TB patients in Bulgaria was estimated to be 10.7% (95% CLs 1.8–44.7) that is higher than in the neighboring countries such as, Romania (2.8% [95% CLs 1.8–4.2]), Greece (1.1% [95% CLs 0.2–7.4]) or Turkey (1.4% [95% CLs 0.2–9.0]) and is more similar to this estimated rate in Ukraine (16% [95% CLs 13.7–18.4]) and Russia (13% [95% CLs 11.3–14.8]) (WHO, 2008a). However one should take notion of the CL values of these estimations. Multiple genes responsible for conferring resistance to the major anti-TB drugs have been identified for M. tuberculosis. A

* Corresponding author. Tel.: +590 590 893881; fax: +590 590 893880. ** Corresponding author. Tel.: +359 2 9793168; fax: +359 2 8700109. E-mail addresses: [email protected] (N. Rastogi), [email protected] (N. Markova). 1567-1348/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.06.006

majority of rifampin (RIF) resistant strains harbor mutations in the 81-bp hot-spot region (rifampin resistance determining region, RRDR) of the rpoB gene encoding DNA-dependent RNA polymerase b-subunit, a target of the drug (Telenti et al., 1993; Ramaswamy and Musser, 1998; Martin and Portaels, 2007). Isoniazid (INH) resistance is controlled by a complex genetic system that involves several genes, katG, inhA, ahpC, kasA, and ndh (Ramaswamy and Musser, 1998; Lee et al., 2001; Martin and Portaels, 2007). Ethambutol (EMB) resistance was most frequently associated with mutations in the embCAB operon which product arabinosyl transferase is involved in mycolic acids metabolism and particularly with mutations in embB codon 306 (Ramaswamy et al., 2000). More recently, Mokrousov et al. (2002b) highlighted a presence of embB306 mutations in EMB-susceptible strains and Hazbo´n et al. (2005) suggested an association of embB306 mutations with broad drug resistance and clustering rather than EMB resistance. In the last decade, the application of different DNA fingerprinting techniques has contributed significantly to our understanding of the transmission of tuberculosis. The most widely applied and standardized molecular typing method for M. tuberculosis complex isolates is IS6110 RFLP typing (van Embden et al., 1993), that is, unfortunately, cumbersome, time-consuming and requires large quantities of DNA. More recently, spoligotyping, a PCR-based reverse-hybridization technique targeting the genetic diversity of the Direct Repeat locus has been proven useful both for molecular epidemiology and evolutionary genetics (Kamerbeek et al., 1997; Brudey et al., 2006). Compared to IS6110 typing, spoligotyping is

V. Valcheva et al. / Infection, Genetics and Evolution 8 (2008) 657–663

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previously (van Embden et al., 1993; Kamerbeek et al., 1997; Supply et al., 2006). The spoligoprofiles were entered into Excel spreadsheets and compared to SITVIT2, an international spoligotype database in Institut Pasteur de Guadeloupe, which is a most recent version of the published SpolDB4 database (Brudey et al., 2006).

more cost-effective, portable, easier to perform, although less discriminating. In recent years, various novel DNA typing methods have been developed. Among them, variable number of tandem repeats (VNTR) typing is probably the most popular approach; the most recently proposed new format for MIRU (mycobacterial interspersed repetitive units)-VNTR typing includes 24 loci (Supply et al., 2006). Characterization of the molecular basis of drug resistance in a survey area constitutes a first step towards an implementation of the methods permitting its fast detection. Here, we report results of the first study on molecular basis of drug resistance in M. tuberculosis strains currently circulating in Bulgaria. We also compared the distribution of the drug resistance in the main genotypic variants defined using spoligotyping and 24-loci MIRUVNTR typing.

2.3. Resistance mutations typing Mutations in rpoB RRDR, katG315, inhA promoter region (positions from 9 to 25), and embB306 were detected as described previously (Morcillo et al., 2002; Mokrousov et al., 2002a,b, 2004, 2006). 2.4. Quality control To minimize the risk of laboratory cross-contamination during PCR amplification, each procedure (preparation of the PCR mixes, the addition of the DNA, the PCR amplification, and the electrophoretic fractionation) was conducted in physically separated rooms. Negative controls (water) were included to control for reagent contamination.

2. Materials and methods 2.1. Study panel The study set comprised 133 M. tuberculosis strains isolated in several provinces across Bulgaria between January 2005 and June 2007; see Supplementary Fig. S1 for details about regions involved and a number of the studied isolates per region. These isolates were recovered from adult pulmonary TB patients who were permanent residents of the country. No preliminary selection of strains based on their drug resistance or patient data was made. All available strains isolated in the mycobacteriology laboratories of the local TB dispensaries were obtained once from each setting (with the exception of Sofia and Haskovo that contributed twice). These isolates corresponded to all newly isolated M. tuberculosis cultures available at the time of collection, hence these clinical isolates may be interpreted as a snapshot of the circulating tubercle bacilli clones in Bulgaria. Susceptibility testing for isoniazid (INH), rifampin (RIF), ethambutol (EMB), streptomycin (STR) was carried out by the absolute concentration method on Lowenstein–Jensen medium as recommended (WHO, 1998). The critical concentrations for INH, RIF, EMB, and SM were 0.25, 10, 2.0, and 4 mg/l, respectively.

2.5. Statistical analysis EpiCalc software was used for calculation of p-values with 95% confidence interval (Gilman and Myatt, 1998). Hunter–Gaston index (HGI) was calculated as described previously (Hunter and Gaston, 1988) and was used to evaluate discriminatory power of the typing methods. PAUP* 4.0 package (Swofford, 2002) was used to reconstruct the most parsimonious dendrogram of the VNTR digital profiles treated as categorical variables. 3. Results The study collection included 37 drug-resistant and 96 susceptible M. tuberculosis strains isolated in different regions of Bulgaria (Supplementary Fig. S1 and Table S1). A monoresistance was identified in 15 of 37 drug-resistant isolates, a majority being limited to the RIF (7/15) and INH (5/15) monoresistance (Table 1). Seventeen strains (12.8%) were resistant to both RIF and INH and thus classified as multidrug resistant. Three types of the rpoB RRDR mutations were found in 20 of 27 RIF-resistant isolates while rpoB S531L (TCG > TTG) was the most

2.2. Genotyping Extraction of DNA from M. tuberculosis strains, spoligotyping and 24-locus MIRU-VNTR typing were performed as described

Table 1 Resistance patterns of 133 M. tuberculosis strains isolated in different regions of Bulgaria No. of isolates (%)

rpoB wild type

rpoB531 TCG > TTG

Resistance type

Phenotypic resistance profilea

MDR

HRc HRE HRS

9 (6.8) 5 (3.7) 3 (2.2)

3 1 1

6 3 2

Polyresistant (non-MDR)

RE HE ES

3 (2.2) 1 (0.8) 1 (0.8)

1 1 1

2

Monoresistant

R H E S

7 5 1 2

1 5 1 2

4

Fully susceptible a b c

(5.3) (3.7) (0.8) (1.5)

96 (72.2)

96

rpoB526 CAC > TAC

rpoB mutant del wt5b

1

katG315 wild type 5 2 1

katG315 AGC > ACC 4 3 2

3 1 1 2

7 3 1 2 96

2

inhA 9 to 25 (wild type) 6 3 3

inhA 15C > T

1

embB306 wild type 6 1 3

2 1 1

2

1 1 1

6 5 1 2

1

7 5

96

One-letter abbreviations of drug resistance: H, INH; R, RIF; E, EMB, S, STR. Absence of hybridization with wild type probe #5, i.e., a mutation in rpoB codons 530–534 (Morcillo et al., 2002; Mokrousov et al., 2006). No information on inhA and embB306 mutations was available for three HR strains.

embB306 mutant

4

2

1 2 96

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frequent (Table 1). The remaining 7 RIF-resistant isolates and all 106 RIF-susceptible isolates had no mutation in the targeted rpoB hot-spot region. Interestingly, 65% (11/17) of MDR isolates were found to harbor a mutation in the rpoB hot-spot region (S531L). Sensitivity and specificity of the genotypic method to detect RIFresistance were 74.1% and 100%, respectively. KatG S315T (AGC > ACC) mutation was detected in 11 (48%) of 23 INH-resistant isolates and in none of 110 INH-susceptible isolates. Additional analysis of the inhA promoter region revealed four isolates that harbored the inhA 15C > T mutation: one INHresistant isolates that also had katG315 mutation and three INHsusceptible isolates. Of these latter, two isolates were RIF- and EMB-resistant (rpoB531 and embB306 mutations), and one isolates was RIF-resistant (rpoB531 mutation). Sensitivity and specificity of the genotypic method to detect INH-resistance were 48% and 100%, respectively. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. No such mutation was detected in EMB-susceptible isolates (Table 1). Sensitivity and specificity of the genotypic method to detect EMB-resistance were 63.6% and 100%, respectively. Strain differentiation was performed by two standardized DNA fingerprinting techniques, spoligotyping and 24-locus MIRU-VNTR typing, in order to assess the relationships among isolates at different levels of genetic relatedness. On the basis of spoligotyping, all 133 isolates were subdivided into 36 distinct spoligotypes (Supplementary Table S2). Twenty spoligotypes represented single isolates; the other 113 isolates were grouped into 16 shared types containing from 2 to 33 isolates. The prevalence of the identified spoligotypes in different regions across the country is shown in Supplementary Table S3. A comparison with SITVIT2 global database permitted us to assign most of the 133 isolates to the known spoligotype families (Supplementary Table S2). Beijing genotype profile (absence of signals from 1 to 34 and presence of at least three of signals from 35 to 43) was not detected in our collection. A selection of 98 isolates belonging to different lineages was subjected to the high-resolution VNTR typing using 24 MIRU-VNTR loci. One should note that a reduction in the sample size did not affect a genetic diversity and representativeness of the studied collection (spoligotyping HGI133 = 0.893 versus HGI98 = 0.912). Previously, we demonstrated a superior discriminatory power of the MIRU-typing over IS6110-RFLP typing for M. tuberculosis strains in Bulgaria (Valcheva et al., 2008a). This explains our choice of 24-loci MIRU-VNTR scheme for a secondary typing in this study. This method differentiated most of the studied isolates (Fig. 1): five shared types consisted of two isolates each, two other shared types consisted of three and four isolates each and the 81 remaining isolates had unique 24-locus digital profiles while HGI was 0.997. Additionally, we defined as single/double locus variation (SDLV) groups those that included isolates differing in one or two loci (Fig. 1). This definition permitted us to identify 19 such groups (shown by grey or black/white circles in Fig. 1) that included from 2 to 6 strains diverging at the cut-off dissimilarity level 0.05. Comparison of the new 15- and 24-locus VNTR formats (Supply et al., 2006) showed that use of the 9 auxiliary loci slightly contributed to the additional differentiation already achieved with the 15-locus scheme by reducing the number of shared types from 10 (22 strains) to 7 (17 strains) and increasing the number of unique patterns from 76 to 81. 4. Discussion Management of tuberculosis is complicated by the emergence of drug-resistant M. tuberculosis strains, which has become a serious health problem worldwide (WHO, 2008a,b). The early

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detection of resistance to first line anti-TB drugs is essential for the efficient treatment and constitutes one of the priorities of TB control of MDR strains. Patients infected with drug-resistant strains are less likely to be cured, and their treatment is more toxic and expensive than the treatment for patients infected with susceptible organisms. Inadequate and/or interrupted therapy allows for the selection of spontaneous mutations in favor of resistant organisms while sequential acquisition of these mutations in different genome loci results in the development of resistance to multiple drugs. Therefore, a correct and rapid detection of resistant strains is necessary for the appropriate and timely anti-TB therapy and the reduction of total treatment cost. In Bulgaria, 1360 TB cases (42% of all new TB cases) were confirmed by culture in 2006; 1108 of them were subjected to DST and 24 (2.2%) of the DST-screened cultures were found to be multidrug resistant (Euro TB, 2008). A total of 22 MDR M. tuberculosis strains were identified in Bulgaria in 2005 (4.6% of all DST-screened cultures from all new TB cases) (Euro TB, 2007). The relative data for 2007 are not yet available. Nevertheless, the extrapolation of the published information (Euro TB, 2007, 2008) allows us to estimate the total number of MDR M. tuberculosis strains identified in Bulgaria between January 2005 and June 2007 (the survey period of this study) to be 58 strains. Consequently, regarding the representativeness of our study panel, we note that the studied sub-sample of the MDR isolates represents 29% (17/58) of the MDR M. tuberculosis cultures isolated from newly diagnosed TB patients in Bulgaria within the survey period. 4.1. Phenotypic versus genotypic drug resistance This study found a high specificity and sufficiently good sensitivity of the molecular methods to detect RIF and EMBresistant strains; the results for INH resistance are more complex. Regarding RIF resistance, the high rate of the rpoB S531L (TCG > TTG) mutation compared to very low rate of the other rpoB mutations found in this study is striking (Table 1). A similar situation was described, e.g., for Russia and Kazakhstan, but it was associated with a Beijing genotype (Mokrousov et al., 2003; Hillemann et al., 2005). In other studies, rpoB 531TTG allele was found in a similar rate of 50% in the Beijing versus non-Beijing RIF-resistant strains from East Asia (Mokrousov et al., 2006; Jou et al., 2005). A variation in the prevalence of this rpoB S531L mutation among Beijing strains in different countries may reflect not only the increased capacity of the Beijing family strains to readily acquire the most frequently observed rpoB mutation but also some specific features of the National TB control programs in different countries (Balabanova et al., 2004; Samarina et al., 2007). In our study the Beijing genotype was not found in Bulgaria among the 133 clinical isolates studied, hence the current situation with MDR-TB in Bulgaria cannot be explained by global dissemination of the Beijing genotype that apparently has not yet reached this country. Whether the very high rate of rpoB S531L mutation correlates with some specific features of the national TB control programs (e.g., a quality of the drugs used) or is hypothetically linked to another molecular mechanism related to acquisition of the RIF resistance needs to be addressed in further investigations in different settings. Molecular investigation of the genetic basis of the INHresistance in M. tuberculosis strains in Bulgaria targeted two the most frequently reported mutations related to INH resistance, katG 315AGC > ACC and inhA 15C > T (Baker et al., 2005; Guo et al., 2006; and references therein). The global prevalence of the katG S315T substitution in INH-resistant strains highlights the selective advantage conferred by this mutation, which appears to provide the optimal balance between decreased catalase activity and a

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Fig. 1. UPGMA dendrogram of M. tuberculosis strains from Bulgaria based on 24 MIRU-VNTR loci. ‘‘SDLV group’’ designates a group of strains differing in single/double locus variation (SLV/DLV). Black and white circles define ‘‘multi-city’’ SDLV groups, grey circles define ‘‘one-city’’ SDLV groups. Loci order in the 24-VNTR loci digital profile: MIRU2,

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sufficiently high level of peroxidase activity in KatG. Mutations in the inhA promoter region are thought to increase the InhA protein expression, thereby elevating the drug target levels and producing INH resistance by a drug titration mechanism (Mdluli et al., 1996). A large-scale study of Hazbo´n et al. (2006) showed that mutations in katG315 were significantly more common in the multidrugresistant isolates while mutations in the inhA promoter were significantly more common in INH-monoresistant isolates. The prevalence of the katG315 AGC > ACC mutation among INHresistant M. tuberculosis strains in the world varies but remains high, e.g., 47% in Finland (Marttila et al., 2008), 61% in China (Jiao et al., 2007), 64% in India (Nusrath Unissa et al., 2008), 93% in Russia (Mokrousov et al., 2002a). Accordingly, it appears that katG S315T mutation alone can be used to reliably predict a high proportion of the INH-resistant strains in many world regions. For Bulgaria this is not the case. Only 11 of 23 INH-resistant strains would be detected genotypically through an analysis of the two targeted mutations and this result is a surprise. Furthermore, inhA mutation was found in only one INH-resistant strain that coharbored a katG315 mutation. Three other strains with inhA mutation were INH-susceptible. It has been suggested that inhA 15C > T mutation can be present by itself and is associated with a low-level INH resistance, 0.2 mg/l (Guo et al., 2006). Hazbo´n et al. (2006) even observed a strong negative association between mutations in katG315 and mutations in the inhA promoter region (p < 0.01). In this study, a MIC was 0.25 mg/l for INH and indeed it may be that the three INH-susceptible strains with inhA promoter mutation (katG315 wild type) had a low-level INH-resistance below the breakpoint used. Perhaps, mutations in other parts of the katG gene and in other gene regions such as, ahpC promoter region, inhA coding sequence or other gene, may account for resistance in other INH-resistant isolates in this study. The results on embB306 variation obtained in this and a recent German study (Plinke et al., 2006) are in line with earlier findings that correlated mutations in embB306 with EMB resistance (Ramaswamy and Musser, 1998; Ramaswamy et al., 2000). They are in contradiction with more recently reported discrepancies between genotypic and phenotypic EMB resistance (Mokrousov et al., 2002b; Hazbo´n et al., 2005, and references therein). A number of explanations of these contradictory findings have been proposed. Plinke et al. (2006) suggested that there is a small difference between the critical concentration used for EMB susceptibility testing and the MIC, making susceptibility testing more problematic. Mokrousov et al. (2002b) hypothesized an unknown mechanism in MDR M. tuberculosis strains that leads to susceptibility to EMB. Hazbo´n et al. (2005) suggested that the clear association between mutations in embB306 and EMB resistance found in several earlier studies might be due to the use of pansusceptible strains as control groups. Regarding this latter point, our study in the Bulgarian setting found embB306 mutation in seven isolates of which six isolates were resistant to more than one drug. Indeed, embB306 mutation was not found in fully susceptible or monoresistant isolates (except for one EMB-monoresistant). However all EMB-susceptible MDR isolates in this study had embB306 wild type allele. Accordingly, it appears that a hypothesis of Hazbo´n et al. (2005) about embB306 as a marker of multidrug resistance is only partly supported by our data. 4.2. Drug resistance versus genotypic variants A specific association of drug resistance properties and particular/predominant clones may be a reason behind a high

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Table 2 Distribution of drug-resistant strains in spoligotypes in this study Spoligotype

All strains

Drug-resistant

MDR

ST125 ST154 ST2905 ST284 ST34 ST4 ST41 ST453 ST47 ST53 Singletons

25 3 5 7 7 5 6 3 7 33 20

3 0 1 3 1 0 3 1 2 11 11

0 0 0 1 0 0 0 1 1 7 7

The total number of strains in this table does not correspond to the total number of strains in this study (n = 133) since the data only on the main shared types (more than two strains) and singletons are shown.

rate of drug resistance (Drobniewski et al., 2005; Narvskaya et al., 2005). We investigated this issue by means of the molecular fingerprinting approach. A detailed analysis of the spoligotype-defined population structure of M. tuberculosis in Bulgaria was presented in our previous publication (Valcheva et al., 2008b). The population structure of M. tuberculosis in Bulgaria appears to be both sufficiently heterogeneous (HGI = 0.893) and dominated by two spoligotypes ST125 (19%) and ST53 (25%). Spoligotype ST53 is found in similar and rather high proportion in the neighboring Greece and Turkey and almost equally distributed across different regions of Bulgaria (Supplementary Table S3). Contrarily, ST125 is not found elsewhere (Valcheva et al., 2008b) and is specific for Bulgaria; furthermore it appears to be mainly confined to the southern part of the country (Supplementary Fig. S1 and Table S3). A comparison with drug resistance data revealed that ST53 and ST125 differ in the rate of MDR/drug-resistant isolates (Table 2). ST125 did not include MDR isolates and the difference in the rate of drug-resistant strains between ST53 and ST125 was noticeable but statistically insignificant (p = 0.11) perhaps due to a small sample size. In may be noted that seventeen MDR isolates exhibited a sufficiently high spoligotype diversity as they represented four shared types (ST53, ST47, ST453 and ST284) and 7 singletons. Furthermore, 11 of 20 spoligotyping-based singletons were drugresistant (Table 2). Taken together, these findings suggest that the emergence and spread of drug-resistant and MDR-TB in Bulgaria are not linked to the spoligotype-defined population structure of M. tuberculosis. Application of the high-resolution 24-locus MIRU typing revealed a significant heterogeneity of our collection. Seven shared types of two to four isolates with identical 24-VNTR profiles have been identified (Fig. 1); they may speculatively correlate with recent transmission/close relatedness of these strains even though an epidemiological link between patients was not established. At the same time, a mathematical modeling of the VNTR loci evolution in M. tuberculosis estimated a very slow mutation rate for the repeats (Grant et al., 2008) and more broadly defined SDLV-groups may reflect a long-term historical evolution of clones. A comparison with city of isolation data revealed that 13 SDLV-groups included isolates from the same city whereas 6 SDLVgroups included isolates from more than one city. The ‘‘one-city groups’’ may speculatively reflect a biologically vertical, family/ household-based transmission that is confined to a particular geographic area. Contrarily, the ‘‘multi-city groups’’ may correlate with a horizontal transmission due to human migration across the

MIRU4, MIRU10, MIRU16, MIRU20, MIRU23, MIRU24, MIRU26, MIRU27 MIRU31, MIRU39, MIRU40, Mtub4, Mtub21, Mtub30, Mtub39, ETRA, ETRC, QUB11b, QUB26, QUB4156, Mtub29, Mtub34, ETRB; ‘‘B’’ means 11 repeat copies in a locus; asterisk (*) designates missing data (PCR failure). Drug resistance: H, INH; R, RIF; E, EMB, S, STR.

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country. Since the first case is somewhat more frequent, a local circulation of particular clones appears to be more significant factor to take into consideration in the molecular epidemiological studies of tuberculosis in Bulgaria. A closer look at the VNTR tree did not identify a genotype or a group of related genotypes associated with drug resistance whereas drug-resistant isolates were evenly dispersed over the dendrogram (Fig. 1). Fifteen of fifty-one SDLV-grouped isolates were drug-resistant; of them 8 were polyresistant. Compared to 12 drug-resistant (8 polyresistant) of the 47 remaining isolates, there was no statistical difference in the prevalence of (multiple) drug resistance between broadly grouped and ungrouped isolates (Fig. 1). 4.3. Concluding remarks A detailed discussion on the reasons of the high MDR-TB rate in both newly and previously diagnosed TB patients in Bulgaria (WHO, 2008a) is beyond the scope of this study. However, it may be noted that a monoresistance, found in 15 of 37 drug-resistant isolates in this study, is known to arise mainly due to noncompliance or wrong prescribing. Accordingly, it may be an additional indication of the somewhat insufficient anti-TB control in Bulgaria. To conclude, rpoB RRDR and embB306 mutations may serve for rapid genotypic detection of the majority of the RIF and EMBresistant M. tuberculosis strains in Bulgaria. The results for INH resistance are complex and further investigation of more genes is needed. A local circulation of the particular clones appears to be an important factor to take into consideration in the molecular epidemiological studies of tuberculosis in Bulgaria. Emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any particular spoligotype or MIRU-VNTR genotype. Acknowledgements We are grateful to all colleagues from regional TB laboratories for kindly providing mycobacterial isolates. This study was supported by NATO’s Public Diplomacy Division in the framework of ‘‘Science for Peace’’ program (grant SFP-982319 ‘‘Detect drugresistant TB’’) and research fellowships from FEMS to Violeta Valcheva and from the European Commission to Igor Mokrousov (Marie Curie Fellowship Contract No. MIF1-CT-2007-039389). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2008.06.006. References Baker, L.V., Brown, T.J., Maxwell, O., Gibson, A.L., Fang, Z., Yates, M.D., Drobniewski, F.A., 2005. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC 46A polymorphism. Antimicrob. Agents Chemother. 49, 1455–1464. Balabanova, Y., Fedorin, I., Kuznetsov, S., Graham, C., Ruddy, M., Atun, R., Coker, R., Drobniewski, F., 2004. Antimicrobial prescribing patterns for respiratory diseases including tuberculosis in Russia: a possible role in drug resistance? J. Antimicrob. Chemother. 54, 673–679. Brudey, K., Driscoll, J.R., Rigouts, L., Prodinger, W.M., Gori, A., Al-Hajoj, S.A., Allix, C., ˜ o, L., Arora, J., Baumanis, V., Binder, L., Cafrune, P., Cataldi, A., Cheong, Aristimun S., Diel, R., Ellermeier, C., Evans, J.T., Fauville-Dufaux, M., Ferdinand, S., Garcia de Viedma, D., Garzelli, C., Gazzola, L., Gomes, H.M., Guttierez, M.C., Hawkey, P.M., van Helden, P.D., Kadival, G.V., Kreiswirth, B.N., Kremer, K., Kubin, M., Kulkarni, S.P., Liens, B., Lillebaek, T., Ho, M.L., Martin, C., Martin, C., Mokrousov, I., Narvskaı¨a, O., Ngeow, Y.F., Naumann, L., Niemann, S., Parwati, I., Rahim, Z., Rasolofo-Razanamparany, V., Rasolonavalona, T., Rossetti, M.L., Ru¨sch-Gerdes, S., Sajduda, A., Samper, S., Shemyakin, I.G., Singh, U.B., Somoskovi, A., Skuce, R.A., van Soolingen, D., Streicher, E.M., Suffys, P.N., Tortoli, E., Tracevska, T., Vincent,

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