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Introduction: Classification of M. tuberculosis complex strains

For a long time the conservation at genome level has prohibited the development of effective molecular methods for the differentiation of clinical M. tuberculosis complex (MTBC) isolates. The differentiation of clinical MTBC isolates was classically based on morphological and phenotypic characteristics such as growth morphology on Löwenstein-Jensen slants (eugonic or dysgonic) or biochemical properties (niacin test, nitrate reduction) (Wayne 1986). Since these tests need sufficient bacterial growth, are time-consuming, do not allow an unambiguous species identification in every case and may not be performed by every laboratory routinely, there was a growing demand for molecular methods that were expected to allow a more rapid and precise species identification.
However, the development of such methods has been hampered by a high similarity on the DNA level and typical targets for species differentiation like the 16S rDNA gene and the 16S-to-23S rDNA internal transcribed spacer are identical (Feizabadi et al. 1996, Frothingham et al. 1994, Niemann et al. 2000, Harmsen et al. 2003).
IS6110 DNA fingerprinting and spoligotyping were among the first methods that have allowed a molecular finer typing of clinical isolates (van Embden et al. 1993, Kamerbeek et al. 1997, van Soolingen et al. 2003). Beyond their intensive use as markers in epidemiological studies, these methods have revealed first insights in the population structure of clinical isolates in different geographical settings. Several genotypes or strain families such as the Beijing genotype have been identified by sharing highly similar IS6110 RFLP and/or characteristic spoligotyping patterns. A large spoligotyping database (SpolDB4) has allowed a first overview on the global population structure and diversity of the MTBC (Brudey et al. 2006). The distribution of particular lineages shows a strong geographical dependence that supports the notion of a phylogeographical population structure and clonal evolution of the MTBC (e.g. van Soolingen et al. 2003, Brudey et al 2006).
Although both methods are well suited for discrimination of clinical isolates they have clear disadvantages for the investigation of the deep phylogenetic structure.
In order to define a population structure more unambiguously, genetic markers need to be independent and, ideally, leading to irreversible events. The establishment of the complete genome sequences of two strains of M. tuberculosis and one strain of M. bovis has allowed a more detailed insight in the genomic structure of MTBC isolates and has facilitated the identification of such markers, namely large sequence polymorphisms (LSPs or RDs; regions of difference) or single nucleotide polymorphisms (SNPs).
A first widely used SNP classification based on SNPs in katG at position 463 and gyrA at position 95 divided the MTBC in three principle genetic groups (Sreevatsan et al. 2007). Thereafter, different RDs have been reported that have been used to define the phylogeny of the M. tuberculosis complex (MTBC) and the identification of particular phylogenetic lineages. Initially, Gordon et al. (1999) proposed a RD classification scheme based on the observation that genomic differences exist between the vaccine strain M. bovis BCG (bacillus Calmette Guérin) and M. tuberculosis H37Rv. These genomic variations (RDs) mainly showed a loss of genetic material in the BCG strain, possibly responsible for its attenuation. Consequently, the genomes of strains of other genotypes were compared and further regions of difference specific for several lineages were discovered (e.g. Gordon et al. 1999, Brosch et al. 2002, Hirsch et al. 2004,). Using primers flanking the particular regions, their presence or absence can easily be determined. Due to the extremely clonal population structure of MTBC and virtually no horizontal gene exchange, such deletions represent robust markers for deep phylogenetic branching and identification of phylogenetic lineages (e.g. Brosch et al. 2002, Hirsch et al. 2004).
In 2002 Brosch et al. proposed a new phylogenetic scenario for the MTBC based on 20 regions of difference with a total sample number of 100 M. tuberculosis isolates. The presence or absence of particular regions was found to be characteristic for several lineages e.g. for M. tuberculosis (TbD1, except EAI strains), M. africanum (RD9), or M. bovis  (RD4). This work was extended by Gagneux and co-workers (2006), who performed a microarray-based investigation of the genome content of 875 MTBC strains from 80 countries. In principle, 6 major lineages have been identified within the MTBC. This population structure was further confirmed by studies applying SNP markers for investigation of the population structure of larger collections of clinical MTBC isolates (for an overview see Gagneux and Small 2007).
However, although both LSPs and SNPs allowed a definition of the main lineages within the MTBC, a finer phylogenetic classification appears to be difficult due to the low variability of such markers in the MTBC clonal complex.
Hence, the use of more variable independent markers for the analysis of the population structure is desirable. Micro- or minisatellites are powerful tools that have been successfully applied for high resolution population-genetics. For the MTBC a typing system based on Variable Number of Tandem Repeats (VNTR)-typing applying genetic elements called Mycobacterial Interspersed Repetitive Units (MIRU) as genetic markers has been proposed as a suitable tool to analyze the genetic diversity of clinical isolates (Supply et al. 2006, Allix et al. 2007). First evaluations already showed that MIRU-VNTR typing can provide unique high-resolution insights into the population structure of the MTBC and provides clear criteria for the identification of the different MTBC lineages and sub-lineages (Supply et al. 2006, Allix et al. 2007).

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