Mycobacterium tuberculosis has a clonal population structure, and the Latin American-Mediterranean (LAM) family is one of the largest and most widespread within this species, showing evidence for remarkable pathobiology and a confusing phylogeny. Here, we applied robust phylogenetic markers to study the evolution of the LAM family and its major sublineages circulating in Russia and neighboring countries. A total of 250 M. tuberculosis isolates were confirmed to belong to the LAM family based on the analysis of the LAM-specific single-nucleotide polymorphisms (SNPs) in the Rv3062 and Rv0129c genes. At this stage, the family status was rectified for 121 isolates misleadingly assigned by CRISPR spoligotyping to non-LAM families (T1- or T5-RUS1). Consequently, the reestimated LAM prevalence rate increased 2-fold in Russia and Kazakhstan and 4-fold in Belarus. The majority (91.8 to 98.7%) of the LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, the Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. Taken together, our findings and further analyses suggest a monophyletic origin of LAM-RUS: at a historically distant time, in Russia, in a small founding bacterial/human population. Its dissemination pattern and high prevalence rate in Northern Eurasia may indicate a long-term coexistence of the LAM-RUS sublineage and local human populations hypothetically leading to coadaptation and reduced pathogenicity of the relatively more ancient clones, such as spoligotype international type 254 (SIT254), compared to the more recent SIT252 and SIT266 clones. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The spread of RD-Rio strains is not as global as commonly claimed and is determined largely by human migration flows (rather than by pathobiological properties of these strains). Consequently, a host population factor appears to play a major role in shaping the in situ dissemination pattern of the imported strains in an autochthonous population.
Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.
Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome.
Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes.
Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.
Mycobacterium tuberculosis; Genome variations; Mutation; Genetic diversity; Whole genome sequencing; Database
The Mycobacterium tuberculosis (MTB) Beijing family isolates are geographically widespread, and there are examples of Beijing isolates that are hypervirulent and associated with drug resistance. One-fourth of Beijing genotype isolates found in Russia belong to the B0/W148 group. The aim of the present study was to investigate features of these endemic strains on a genomic level. Four Russian clinical isolates of this group were sequenced, and the data obtained was compared with published sequences of various MTB strain genomes, including genome of strain W-148 of the same B0/W148 group. The comparison of the W-148 and H37Rv genomes revealed two independent inversions of large segments of the chromosome. The same inversions were found in one of the studied strains after deep sequencing using both the fragment and mate-paired libraries. Additionally, inversions were confirmed by RFLP hybridization analysis. The discovered rearrangements were verified by PCR in all four newly sequenced strains in the study and in four additional strains of the same Beijing B0/W148 group. The other 32 MTB strains from different phylogenetic lineages were tested and revealed no inversions. We suggest that the initial largest inversion changed the orientation of the three megabase (Mb) segment of the chromosome, and the second one occurred in the previously inverted region and partly restored the orientation of the 2.1 Mb inner segment of the region. This is another remarkable example of genomic rearrangements in the MTB in addition to the recently published of large-scale duplications. The described cases suggest that large-scale genomic rearrangements in the currently circulating MTB isolates may occur more frequently than previously considered, and we hope that further studies will help to determine the exact mechanism of such events.
We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a “successful” clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.
The Beijing genotype is a globally spread lineage of Mycobacterium tuberculosis. In Russia, these strains constitute half of the local population of M. tuberculosis; they are associated with multidrug resistance and show increased transmissibility. Here, we analyzed traditional and new markers for the rapid and simple genotyping of the Beijing strains. A representative sample of 120 Beijing genotype strains was selected from a local IS6110-restriction fragment length (RFLP) database at the St. Petersburg Pasteur Institute. These strains were subjected to variable-number tandem-repeat (VNTR) typing using 24 loci of a newly proposed format and three hypervariable (HV) loci (QUB-3232, VNTR-3820, and VNTR-4120). Ten of the 27 VNTR loci were monomorphic, while five loci, MIRU26, QUB-26, QUB-3232, VNTR-3820, and VNTR-4120, were the most polymorphic (Hunter Gaston index, >0.5). VNTR typing allowed us to differentiate between two large IS6110-RFLP clusters known to be prevalent across the entire country (clusters B0/W148 and A0) and identified in 27 and 23% of strains, respectively, in the Beijing genotype database. The B0/W148 strains were grouped closely in the VNTR dendrogram and could be distinguished by a characteristic signature of the loci MIRU26 and QUB-26. Consequently, this clinically important IS6110-RFLP variant, B0/W148, likely presents a successful clonal group within the M. tuberculosis Beijing lineage that is widespread in Russia. To conclude, the IS6110-RFLP method and VNTR typing using a reduced set of the most polymorphic loci complement each other for the high-resolution epidemiological typing of the M. tuberculosis Beijing genotype strains circulating in or imported from Russia.
The present study evaluated new markers for molecular typing of Mycobacterium tuberculosis with a collection of strains circulating in Bulgaria. A study sample included 133 strains from epidemiologically unlinked patients from different regions of the country. Spoligotyping was used as a primary typing tool; it subdivided these strains into 37 types, including 15 clusters and 22 singletons. Traditional IS6110-restriction fragment length polymorphism (RFLP) typing and novel 24-locus variable number tandem-repeat (VNTR) typing methods were applied to the selection of 73 strains. Discriminatory power (Hunter-Gaston index [HGI]) of these methods was found to be 0.983 and 0.997, respectively. The 73 strains were subdivided into 66 types by a 24-locus mycobacterial interspersed repetitive unit (MIRU)-VNTR scheme, 62 types by a classical 12-locus MIRU-VNTR scheme, 51 types by IS6110-RFLP typing, and 31 types by spoligotyping. A combination of the five most polymorphic loci (MIRU40, Mtub04, Mtub21, QUB-11b, and QUB-26) was shown to achieve a high discrimination (HGI = 0.984). To conclude, a complete 24-locus scheme excellently differentiated strains in our study, whereas a reduced 5-locus set provided a sufficiently high differentiation and may be preliminarily suggested for the first-line typing of M. tuberculosis isolates in Bulgaria.
In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin- and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.
The newly proposed variable-number tandem-repeat (VNTR) typing system, which includes a basic 15-locus set and a high-resolution 24-locus set (P. Supply et al., J. Clin. Microbiol. 44:4498-4510, 2006), demonstrated a high power for the discrimination of Mycobacterium tuberculosis isolates collected worldwide. To evaluate its ability to differentiate the Beijing genotype strains from the Beijing area in China, 72 isolates with typical Beijing or Beijing-like spacer oligonucleotide typing profiles were subjected to typing with the VNTR system (24 loci) and typing by restriction fragment polymorphism analysis with IS6110 (IS6110-RFLP). Compared to the “old” 12-locus VNTR typing method, use of the 15- and 24-locus systems had a dramatically improved power to discriminate the Beijing genotype strains. A subtle difference in the Hunter-Gaston discriminatory index (HGI) between the 15-locus and the 24-locus systems resulted from only one locus, Mtub29. However, the VNTR-based clusters could be further differentiated by IS6110-RFLP (HGI by IS6110 RFLP, 0.999), although in one case an IS6110 cluster was subdivided by the 15-locus VNTR system. In this sense, use of the newly proposed 15-locus VNTR system along with the Mtub29 locus can serve as a first-line typing method for the epidemiological study of M. tuberculosis isolates in Beijing, while secondary typing of clustered strains by IS6110-RFLP is still required.
Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.
The Mycobacterium tuberculosis Beijing genotype strains appear to be hypervirulent and associated with multidrug-resistant tuberculosis. Therefore, the development of a both rapid and simple method to detect the M. tuberculosis Beijing genotype is of clinical interest per se. Previously, we described a simple and fast approach to detect the Beijing genotype based on IS6110 inverse-PCR typing. Here, we evaluated this method against a large, diverse, and recent collection of strains. The study sample included 866 M. tuberculosis strains representing but not limited to the regions in Russia, Europe, and East Asia where the Beijing genotype is endemic. Based on a spoligotyping method, 408 strains were identified as Beijing genotypes; they were additionally subdivided into ancient and modern sublineages based on the analysis of the NTF locus. All strains were further subjected to the IS6110-based inverse PCR. All of the Beijing genotype strains were found to have identical two-band (ancient sublineage) or three-band (modern sublineage) profiles that were easily recognizable and distinct from the profiles of the non-Beijing strains. Therefore, we suggest using IS6110-based inverse-PCR typing for the correct identification of the Beijing genotype and its major sublineages. The method is fast and inexpensive and does not require additional experiments but instead is implemented in the routine typing method of M. tuberculosis.
We compared the population structure and drug resistance patterns of the Mycobacterium tuberculosis strains currently circulating in the Beijing area of China. One hundred thirteen of 123 strains belonged to the Beijing family genotypes defined by spoligotyping. The Beijing genotype strains were further subdivided into old and modern sublineages on the basis of NTF locus analysis. A stronger association with resistance to the more recently introduced antituberculosis drugs has been observed for old versus modern strains of the Beijing genotype, suggesting that its different sublineages may differ in their mechanisms of adaptation to drug selective pressure.
A large diphtheria epidemic in the 1990s in Russia and neighboring countries was caused by a clonal group of closely related Corynebacterium diphtheriae strains (ribotypes Sankt-Peterburg and Rossija). In the recently published complete genome sequence of C. diphtheriae strain NCTC13129, representative of the epidemic clone (A. M. Cerdeño-Tarraga et al., Nucleic Acids Res. 31:6516-6523, 2003), we identified in silico two direct repeat (DR) loci 39 kb downstream and 180 kb upstream of the oriC region, consisting of minisatellite (27- to 36-bp) alternating DRs and variable spacers. We designated these loci DRA and DRB, respectively. A reverse-hybridization macroarray-based method has been developed to study polymorphism (the presence or absence of 21 different spacers) in the larger DRB locus. We name it spoligotyping (spacer oligonucleotide typing), analogously to a similar method of Mycobacterium tuberculosis genotyping. The method was evaluated with 154 clinical strains of the C. diphtheriae epidemic clone from the St. Petersburg area in Russia from 1997 to 2002. By comparison with the international ribotype database (Institut Pasteur, Paris, France), these strains were previously identified as belonging to ribotypes Sankt-Peterburg (n = 79) and Rossija (n = 75). The 154 strains were subdivided into 34 spoligotypes: 14 unique strains and 20 types shared by 2 to 46 strains; the Hunter Gaston discriminatory index (HGDI) was 0.85. DRB locus-based spoligotyping allows fast and efficient discrimination within the C. diphtheriae epidemic clonal group and is applicable to both epidemiological investigations and phylogenetic reconstruction. The results are easy to interpret and can be presented and stored in a user-friendly digital database (Excel file), allowing rapid type determination of new strains.
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).
We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.
The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.
We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.
Mycobacterium tuberculosis; spoligotyping
A total of 183 epidemiologically unlinked Mycobacterium tuberculosis isolates collected in the St. Petersburg area of Russia from 1996 to 2001 were screened for alterations in codon 306 of the embB gene; mutations in this codon are reported to confer resistance to ethambutol (EMB). The embB306 mutations were detected in 14 (48.3%) of 29 EMB-resistant strains and, quite surprisingly, in 48 (31.2%) of 154 EMB-susceptible strains. A discrepancy between the results of phenotypic and genotypic EMB resistance tests was restricted to the strains already resistant to other antitubercular (anti-TB) drugs. In particular, 40 (60%) of the 69 EMB-susceptible strains resistant to rifampin, isoniazid, and streptomycin but none of the 43 pansusceptible strains harbored an embB306 mutation. We hypothesize that the phenomenon observed could reflect the presence of a target other than EmbB for the drug in tubercle bacilli; this unknown target could be sensitized and affected, sensu lato, by EMB during treatment with other first-line anti-TB drugs. Comparison with DNA fingerprinting data showed that, irrespectively of the phenotypic susceptibility profiles, 46 (50.6%) of 91 Beijing family strains and 16 (17.4%) of 92 strains of other genotypes had a mutation in embB306.
We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC→ACC and ACA (Ser→Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inhr strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.
A total of 204 isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different patients in the northwestern region of Russia from 1996 to 2001 were screened by a PCR-restriction fragment length polymorphism (RFLP) assay. This assay uses HapII cleavage of an amplified fragment of the katG gene to detect the transversion 315AGC→ACC (Ser→Thr), which is associated with INH resistance. This analysis revealed a 93.6% prevalence of the katG S315T mutation in strains from patients with both newly and previously diagnosed cases of tuberculosis (TB). This mutation was not found in any of 57 INH-susceptible isolates included in the study. The specificity of the assay was 100%; all isolates that contained the S315T mutation were classified as resistant by a culture-based susceptibility testing method. The Beijing genotype, defined by IS6110-RFLP analysis and the spacer oligonucleotide typing (spoligotyping) method, was found in 60.3% of the INH-resistant strains studied. The katG S315T shift was more prevalent among Beijing genotype strains than among non-Beijing genotype strains: 97.8 versus 84.6%, respectively, for all isolates, including those from patients with new and previously diagnosed cases, isolated from 1999 to 2001 and 100.0 versus 86.5%, respectively, for isolates from patients with new cases isolated from 1996 to 2001. The design of this PCR-RFLP assay allows the rapid and unambiguous identification of the katG 315ACC mutant allele. The simplicity of the assay permits its implementation into routine practice in clinical microbiology laboratories in regions with a high incidence of TB where this mutation is predominant, including northwestern Russia.