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Journal of Clinical Microbiology, June 2004, p. 2438-2444, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2438-2444.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of the Allelic Diversity of the Mycobacterial Interspersed Repetitive Units in Mycobacterium tuberculosis Strains of the Beijing Family: Practical Implications and Evolutionary Considerations

Igor Mokrousov,^1* Olga Narvskaya,¹ Elena Limeschenko,¹ Anna Vyazovaya,¹ Tatiana Otten,² and Boris Vyshnevskiy²

Laboratory of Molecular Microbiology, St. Petersburg Pasteur Institute, 197101 St. Petersburg,¹ Laboratory of Microbiology of Tuberculosis, The Research Institute of Phthisiopulmonology, 193063 St. Petersburg, Russia²

Received 26 September 2003/ Returned for modification 19 November 2003/ Accepted 5 February 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
A study set comprised 44 Mycobacterium tuberculosis strains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mycobacterium tuberculosis is justly considered one of the most successful human pathogens; it infects one-third of the human population and kills about 3 million people every year (4). The population structure of this species appears to be strictly, or at least predominantly, clonal; this has been demonstrated by utilizing different molecular markers and study designs (1, 16, 25, 28). Several genetic families have been identified within this biological species (e.g., Haarlem, Beijing, and East-African-Indian) (23, 33). Most likely, these families could have initially been endemic within specific geographical areas. Some of these remain circumscribed to the particular regions, whereas others have become omnipresent. The Beijing family is an example of the latter group. This genetic family, first identified in 1992 in Beijing, China (33), is currently almost omnipresent and significantly prevalent (up to 40 to 60%) in certain world regions, e.g., East Asia and the former USSR (2, 5, 13, 17). Genetically, these strains are closely related and thus difficult to differentiate by most of the currently used typing techniques (2, 5). The "gold standard" method to identify Beijing strains is spoligotyping (absence of signals 1 to 34 [2, 5, 15]), whereas the best approach to further subtype them is still IS6110-restriction fragment length polymorphism (RFLP) typing (33). This method is rather time-consuming and cumbersome, since it requires extraction and purification of large amounts of DNA, and hence a simpler (and reliable) alternative typing method is needed. The recently introduced mycobacterial interspersed repetitive units (MIRU) typing is based on size analysis of the PCR-amplified variable number of tandem repeats (VNTR) loci, and it requires only basic PCR and agarose electrophoresis equipment (26, 27, 29). This new method was shown with different strain samples to possess a higher discriminatory power than that of spoligotyping and only slightly below that of IS6110-RFLP typing (29). The apparent advantage of the MIRU approach (compared to the IS6110 typing) is its portability due to easy digitalization of the generated profiles and hence easy interlaboratory exchange, as well as easy creation and maintenance of the databases.

In the present study, we evaluated the discriminatory ability of the MIRU typing on the representative set of different Beijing strains circulating in Russia. Further, we compared our results against published MIRU profiles of the Beijing strains from other geographical sites attempting to gain some insights into the global phylogeny of this important genetic family within M. tuberculosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. M. tuberculosis strains were recovered from adult patients with pulmonary tuberculosis. These patients came from St. Petersburg and other regions of the Russian Federation and were admitted to the hospitals of St. Petersburg Research Institute of Phthisiopulmonology and City Anti-Tuberculosis Dispensary of St. Petersburg between 1996 and 2003.

DNA fingerprinting. The DNA of the studied strains was isolated according to the recommended method (32) and subjected to IS6110-RFLP typing and spoligotyping as described previously (7, 32). The IS6110-RFLP patterns were compared by using GelCompar version 4.1 package (BVBA Applied Maths, St. Martens-Latem, Belgium) by unweighted pair-group method of arithmetic averages (UPGMA) by using the Dice coefficient.

MIRU analysis was performed essentially as described by Supply et al. (29) with Tth polymerase (Eurobio) for the PCR. The amplicons were evaluated on the 1.5% standard (Quantum) agarose gels with a 100-bp DNA ladder (Amersham Bioscience). The H37Rv strain was run as additional control of the performance of the method in our laboratory. Search of the MIRU profiles of the Beijing strains from other studies was done by using the Pubmed-Entrez and Google engines, followed by inspection of the retrieved articles for the presence of the information on Beijing isolates. The copy numbers in each of the 12 MIRU loci were converted into eight-digit codes where the presence or absence of one copy in the repeat was represented as 1 or 0 (e.g., three copies: 1110000). A similar coding system treating VNTR data as continuous variables was previously used for ETR-VNTR analysis (23). We used an eight-digit code because a maximal copy number in one MIRU locus was eight in our sample. The resulting 96-character binary matrix was used to construct the UPGMA-based tree (1-Jaccard coefficient) by using Recogniser software of the Taxotron package (6).

Statistical analysis. The Hunter-Gaston discriminatory index (HGDI) was calculated as described previously (11). The mean HGDI was calculated as a mean value of the HGDIs for 12 particular MIRU loci.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 198 Beijing family strains (of 354 M. tuberculosis strains studied) from epidemiologically unlinked patients (Russian Federation, 1996 to 2003) have previously been identified based on their specific spoligotyping signature (absence of spacers 1 to 34) and by IS6110-RFLP typing (14, 17; O. Narvskaya et al., unpublished data). Most of the Beijing strains (n = 193) had a nine-signal spoligoprofile (signals 35 to 43) and similar IS6110-RFLP profiles characteristic of the Beijing genotype (2). Three strains had Beijing characteristic IS6110-RFLP profiles and incomplete Beijing-like spoligoprofiles disrupted in particular signals (37, 37 and 38, and 40 [12]). In addition, two strains with the nine-signal Beijing spoligoprofile had IS6110-RFLP profiles (12 and 14 copies of IS6110) more distant from those of the other 196 strains (similarity of 60 to 70%%). These two strains were previously designated "atypical" and were thought to be ancient Beijing strains based on the use of other molecular markers (IS1547 and Rv3135-PPE [13]).

For the present study, we selected 44 strains representing the overall diversity of the Russian Beijing IS6110-RFLPs and spoligoprofiles, including both ancient Beijing strains and two strains with Beijing-like spoligoprofiles (37 and 40) (Fig. 1) and evaluated the MIRU-VNTR method (29) to differentiate them. Based on the use of all 12 MIRU loci taken together, a total of 12 types were identified and designated M1 to M12. Two large distinct clusters included 24 (M2) and 10 (M11) identical strains, whereas other isolates demonstrated unique combinations of the 12-MIRU signatures (Table 1). The M2 type included isolates with clearly different IS6110-RFLP profiles, distributed equally on all branches of the IS6110-based tree (compare, for example, strains 6358, 7315, and 7091 in Fig. 1). On the other hand, the M11 type included a closely related group of nine strains and one ancient Beijing strain 2069 (Fig. 1). The other ancient Beijing strain (strain 3242), the most distant from all other strains on IS6110-RFLP based tree, exhibited a unique MIRU profile (M12).

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FIG. 1. IS6110-RFLP-based UPGMA tree of the representative sample of Russian Beijing strains. Superscript letters: a, ancient Beijing strains (13); b, Beijing strains with disrupted spoligoprofiles in single signals, 37 or 40; c, B/W148 IS6110-RFLP profile (2, 18).

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TABLE 1. MIRU-VNTR profiles of M. tuberculosis Beijing strains from different areasa

HGDI scores were calculated for particular MIRU loci for the Russian Beijing strains (Table 2). Application of the single MIRUs provided poor discrimination of the different Russian Beijing strains, with the two largest clusters encompassing more than 90% of the strains (Table 2). Most of the loci were monomorphic (loci 2, 4, 23, 24, 27, and 39) or almost monomorphic (loci 10, 16, 20, and 40: 0 < HGDI < 0.1). The best discrimination with a single MIRU was achieved with MIRU26 (four alleles, HGDI = 0.49, Table 2) and, to a much lesser degree, with MIRU31 (three alleles, HGDI = 0.20). The HGDI values for all 12 loci used together and for a combination of the loci 26 + 31 were 0.65 and 0.57, respectively. Most strains with seven copies of MIRU26 belonged to the same MIRU type (M11) and IS6110-RFLP cluster (asterisk in Fig. 1).

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TABLE 2. Discriminatory power of the 12 MIRU loci used alone and together to differentiate M. tuberculosis Beijing strains in Russian, South African, and global settingsa

We have searched for published MIRU profiles of Beijing strains from other studies carried out in different world regions. The information on the total of 54 strains was found (3, 8, 24, 28, 29), including three additional Russian strains, while the major non-Russian samples were South African (n = 38) and East Asian (n = 7) strains. Thus, further analysis was done for 98 strains (a complete table of their MIRU profiles is available upon request).

Since the major strain groups represented Russian (n = 47) and South African (n = 38) strains, a detailed analysis was done for Russian, South African, and global settings. Phylogenetic analysis of the 98 strains identified 32 MIRU types that comprised nine clusters and 23 unique isolates (Table 2 and Fig. 2). Several distinct groups of closely related types can be identified in the dendrogram (Fig. 2). The HGDI value was highest for the global set of Beijing strains: 0.86 versus 0.75 and 0.65 for South African and Russian settings, respectively (Table 2). A higher diversity of the South African MIRU profiles is demonstrated as the increased allelic variation of the particular loci (i.e., loci 4, 10, 16, 27, and 39) that had more number of alleles compared to Russian Beijing strains (four versus three or two alleles; Table 2). We also note a marked difference in the allelic diversity of certain loci (i.e., loci 4, 10, 26, and 39) in Russian and South African Beijing strains. In particular, locus 26 had an HGDI value in Beijing strains in Russian, South African, and global settings of 0.49, 0.25, and 0.55, respectively. Several classifications of the MIRU loci have recently been proposed (24, 29). In particular, Sola et al. (24) suggested, based on HGDI, to consider the MIRU loci as highly (>0.6), moderately (0.3 to 0.6), and poorly (<0.3) discriminating. When this finding is applied to our results, five loci show negligible diversity (HGDI < 0.1, Table 2) and only locus 26 in the Russian Beijing and loci 10 and 39 in the South African Beijing strains provide a moderate discrimination level. The mean HGDI scores were 0.08 and 0.17 for Russian and South African Beijing samples, respectively. The best minimal combination among all tested combinations (not shown) to differentiate Beijing strains was found to be three MIRU loci 10 + 26 + 39 for South African (HGDI 0.66) and global settings (0.76), and a combination of loci 26 + 31 for Russian Beijing strains, as mentioned above.

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FIG. 2. UPGMA tree of the MIRU types identified in Beijing strains from different geographical locations. Types including more than one strain are in boldface; large types (>10 strains) are in shaded boxes. Strain origins: MNG, Mongolia; RUS, Russia; CHN, China; MYS, Malaysia ZAF, South Africa; KOR, Korea; THA, Thailand; CAF, Central African Republic; GUF, French Guiana.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The M. tuberculosis Beijing strains attract great attention worldwide since they demonstrate some important pathogenic features, such as increased virulence in BCG-vaccinated mice (10), association with multiple-drug resistance (30), increased risk of febrile response in patients (31), ability to more rapidly multiply in human macrophages (34), and presumably easier adaptation to the changing environment due to mutator alleles of the mutT genes (19). Therefore, the ongoing global dissemination of these strains threatens the success of national tuberculosis control programs. Molecular epidemiological investigations involving Beijing strains are complicated by their considerable genetic homogeneity (2, 5). In practice, this means that in certain areas (such as East Asia and the former USSR), where half of the circulating strains belong to the genotype, they are virtually untypeable since they cannot be differentiated by most typing methods except for IS6110-RFLP. We have undertaken the present study in order to assess the possibility to differentiate Russian Beijing strains by the recently developed MIRU method based on the analysis of 12 MIRU-VNTR loci (29). We further compared our results with published MIRU profiles of Beijing strains from other areas in order to assess a geographic variation of the MIRU loci in Beijing strains and to gain some insights into the specific features of their global evolutionary relationships.

Our results clearly demonstrate that the MIRU method (29) cannot substitute IS6110-RFLP analysis for comprehensive epidemiological subtyping of the Beijing strains in Russia. The set under study included 44 Russian Beijing strains with different IS6110-RFLP profiles (Fig. 1). Most of these strains were, however, identical in the composition of all 12 MIRU loci and comprised two types, M2 and M11, encompassing 24 and 10 strains, respectively (Fig. 1). At the same time, the MIRU PCR based method requiring only small quantities of DNA can be utilized for rapid rough subdivision of the Beijing strains. Calculation and comparison of the HGDI values (Table 2) reveals several possibilities to apply MIRUs for analysis of these strains. First, in the case of analysis of a local population of the Beijing strains, a limited number of loci can be used, such as the combination 26 + 31 (HGDI = 0.57) or just 26 (HGDI = 0.49) in the Russian setting (HGDI_12-loci 0.65). In the South African setting, more MIRU loci were slightly more polymorphic in Beijing strains, and therefore more combinations can be used, such as 4 + 10 + 39 (HGDI = 0.63) or 10 + 26 + 39 (0.66), compared to HGDI for all 12 loci (0.75). Second, a larger number of loci may eventually be evaluated to distinguish a suspect imported Beijing isolate from the indigenous strains, whereas the loci found to be (almost) monomorphic in all Beijing strains studied (loci 2, 20, 23, and 24; Table 2) may be excluded from epidemiological investigation. Third (and ideally), the evaluation of all 12 loci (including the monomorphic ones) in all Beijing strains is still obligatory for theoretical purposes in order to uncover a marginal variation and trace minor variants and finally to update the global MIRU database of M. tuberculosis. For example, despite generally lower diversity of Russian Beijing strains and a larger number of completely monomorphic loci compared to the South African Beijing strains, a variant allele of the MIRU20 was found in one Russian Beijing strain (one copy versus the usual two copies).

The apparently low diversity of the MIRU loci in Beijing strains allowed us to use the available data for a phylogenetic analysis. Previously, we proposed a recent dissemination of the circulating Beijing clone in Russia (95% of Russian Beijing strains [13]). Presently, we conclude that two sublineages appear to shape this process: the first one includes type M11 (10 strains) and 3 single isolates (types M1, M8, and M9). The second sublineage includes type M2 (27 strains) and 5 isolates (types M4, M5, M7, and M10). Finally, two strains (3242 and 2069, Fig. 1) representing a small proportion of the Russian Beijing strains (<5%) were previously suggested to be ancient Beijing strains (13). It was interesting to see their position under the MIRU analysis: whereas the ancient strain 3242 had indeed a quite distinct MIRU signature (M12), surprisingly, the other ancient Beijing strain 2069 belonged to the large type of "modern" Beijing strains (M11). Furthermore, this intriguing type M11 includes also strains from other areas and Russian strains with IS6110-RFLP profile W148/B (Fig. 1), the most frequent Russian Beijing variant identified in approximately one-third of the Russian Beijing strains (2, 18). We hypothesize that type M11 could have resulted from a convergent evolution due to a possible biological role of some MIRUs (21, 26, 27) or could present a stable conserved combination achieved long ago and unchanged since evolutionary distant time.

Comparison of the MIRU diversity in South African versus Russian Beijing samples reveals: (i) more copies in the most "Beijing-discriminating" locus MIRU26 (generally, seven or eight versus five or seven; Table 1), (ii) a smaller number of completely monomorphic loci (four versus six; Table 2), and (iii) higher mean HGDI scores (0.17 versus 0.08). If we assume that more diversity is generated due to longer evolutionary history (clonal expansion), then, taken together, these findings might reflect a more recent dissemination of the currently circulating and locally predominant Beijing strains in South Africa compared to Russia.

Analysis of the global tree of the Beijing strains from different geographic regions indicates a clear separation of Russian from South African strains (Fig. 2). These strains presented two the most numerous samples in our analysis; however, they dropped to the different MIRU types. Unlike major Russian types, none of the large South African types included strains from other locations. Furthermore, South African types were not shared by any other Beijing strains though South African types were situated well "inside" the global MIRU-based tree. In principle, the latter observation illustrates a monophyly of the Beijing family as a whole. The situation with the South African Beijing strains may be explained in terms of the strictly clonal evolution of the M. tuberculosis strains in general and that of the Beijing strains, in particular, which is currently uninfluenced by any significant horizontal gene transfer although analysis of additional strains from Asia and Americas is needed to confirm our conclusions.

To sum up, we suggest a stepwise scheme involving the MIRU typing in order to analyze an M. tuberculosis Beijing isolate: (i) identification by spoligotyping or other rapid method (e.g., inverse IS6110 PCR [15]), (ii) preliminary differentiation by using two or three specific MIRUs (e.g., 26 + 31 and 10 + 26 + 39 for Russian and South African settings, respectively), and (iii) complete 12-MIRU-loci typing, and IS6110-RFLP analysis. A preliminary evaluation is necessary for each geographical site to assess the diversity of particular loci in local Beijing strains prior to implementation of the MIRU method into routine use. Perhaps inclusion of other VNTR loci (9, 20, 21, 22) would aid in the better discrimination of Beijing strains.

 
We thank Lidia Steklova for providing some of the clinical isolates and Alessandra Riva for critical reading of the manuscript and language corrections. We are grateful to authors of several articles (3, 8, 24, 28, 29), whose results were used in this study. We also thank two anonymous reviewers for their valuable comments and suggestions.

This study received partial support from Institut Pasteur, Paris, France, and Louis D. Award of the French Academy of Sciences.

 
* Corresponding author. Mailing address: Pasteur Institute, 14, Mira St., St. Petersburg 197101, Russia. Phone: 7-812-233-21-49. Fax: 7-812-232-92-17. E-mail: miv{at}IM4520.spb.edu.

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Journal of Clinical Microbiology, June 2004, p. 2438-2444, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2438-2444.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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