Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ϳ30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RD Rio . Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RD Rio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RD Rio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RD Rio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RD Rio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RD Rio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RD Rio M. tuberculosis.Tuberculosis (TB) is a preventable and curable infectious disease that nonetheless remains a significant cause of morbidity and mortality in resource-poor nations (14). TB also threatens to reemerge in developed nations as a consequence of increased immigration, its synergy with the human immunodeficiency virus (HIV)/AIDS epidemic, and a deprioritization of TB control efforts in public health policy (64). The principal etiologic agent of human TB is Mycobacterium tuberculosis, but other species within the M. tuberculosis complex (MTC), such as "Mycobacterium canettii" (proposed name), Mycobacterium africanum, and Mycobacterium bovis, are also known to cause TB in humans (30). Although the MTC is considered a relatively homogenous taxon at the DNA sequence level, an increasing number of species-, lineage-, and strain-specific genetic variations have been revealed by the identification of multicopy repeat elements, targeted interrogations of specific genetic loci, and the comparison of whole-genome sequences of several MTC species and strains (21,24,31). These differences have been exploited as markers for epidemiological purposes and/or to assist in the identific...

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

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MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil. Key words: Mycobacterium bovis -spoligotyping -MIRU-VNTR typing -bovine tuberculosis -molecular epidemiologyGenotyping of M. bovis from Brazil • Patrícia Martins Parreiras et al. 65While IS6110-RFLP lacks resolution in this species, spoligotyping has been demonstrated to be a fast and cost-effective method for first-line typing (Haddad et al. 2004). The MIRU-VNTR typing procedure has been extensively evaluated in M. tuberculosis and the recent data available about this method on M. bovis strains confirmed its considerable power of discrimination (Sola et al. 2003, Michel et al. 2008, 2010. Smith et al. (2006) inferred that a combination of spoligotyping and VNTR typing resulted in the simplest and most cost-effective method for routine molecular typing and epidemiological tracing of bovine TB in Great Britain.Some previous studies have successfully employed techniques such as IS6110-RFLP and spoligotyping to genotype M. bovis strains isolated from cattle in Brazil (Zanini et al. 2001, Rodriguez et al. 2004), but as far as we know, no data exist on genotyping of M. bovis by combining spoligotyping and MIRU-VNTR typing in our country. We aimed to use spoligotyping and MIRU-VNTR typing to assess the genetic diversity of Brazilian M. bovis isolates. MATERIALS AND METHODS Sampling and conventional procedures -During 2001and 2002, tissue samples from cattle with lesions suggestive of TB were collected immediately after slaughter at the time of inspection in abattoirs. The study samples were collected in abattoirs that followed slaughter inspection procedures from the states of Amazonas (AM), Paraíba (PB), Distrito Federal (DF), Mato Grosso do Sul (MS), São Paulo, Santa Catarina (SC) and Minas Gerais (MG). The sampled specimens from respective carcasses were pooled, put on ice and sent to the Laboratório Nacional Agropecuário de Minas Gerais (Pedro Leopoldo, MG), where they were processed for inoculation in Stonebrink culture medium (Mota 1985). The cultures were incubated at 37ºC and verified for bacterial growth every week for a period of at least two months. Bacterial isolates were submitted to standard procedures for differentiation of certain species of the MTBC, including the evaluation of niacin production, nitrate reductase activity, Tween hydrolysis, catalase activity at 68ºC and resis...

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Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.