Ecotypes of the Mycobacterium tuberculosis complex
Introduction
To further understand the nature and evolution of bacterial species we have investigated the animal-adapted strains of the Mycobacterium tuberculosis complex. This group of bacteria are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA sequences and include the important human pathogen M. tuberculosis (Sreevatsan et al., 1997; Brosch et al., 2002). The population structure of this group of organisms is apparently highly clonal, with no recombination of chromosomal sequences between strains (Cole et al., 1998; Gutacker et al., 2002; Hirsh et al., 2004). In the absence of inter-strain recombination once a non-repetitive chromosomal region has been deleted it cannot be replaced and the deletion is a marker of a single cell and all its descendants. This feature of a clonal organism was exploited by Brosch et al. (Brosch et al., 2002) to describe a new evolutionary scenario for this group which concluded that all of the strains host-adapted to animals formed a nested lineage marked by the absence of a specific chromosomal region (RD9). The RD9 deleted lineage, which excludes the human-adapted pathogen M. tuberculosis as well as the more distantly related M. canettii, includes strains of an apparently human-adapted species, M. africanum, as well as strains adapted to a variety of other mammals including M. microti (found in voles, wood mice and shrews), M. pinnipedii (found in marine mammals), M. caprae (associated with goats) and M. bovis (associated with cattle). The lineage also includes strains, such as the Oryx strain, that have not been given a species name but are identified by the host from which they are most frequently recovered (Lomme et al., 1976).
The most common molecular typing method applied to animal-adapted strains of the M. tuberculosis complex is spoligotyping (Kamerbeek et al., 1997; Durr et al., 2000). This molecular typing method identifies polymorphism in the presence of spacer units in the direct repeat region in strains of the M. tuberculosis complex (Kamerbeek et al., 1997; van der Zanden et al., 1998). The direct repeat region is composed of multiple, virtually identical, 36-bp repeats interspersed with a series of unique spacer sequences of similar size. Spoligotype patterns are polymorphic among isolates due to the absence of one or more spacers and adjacent repeat units. Several studies of this region in strains of M. tuberculosis have concluded that the evolutionary trend of this region is primarily by loss of one or several contiguous spacer regions (Groenen et al., 1993; Fang et al., 1998; van Embden et al., 2000). We have investigated the loss of spoligotype spacer units in strains of the RD9 deleted lineage described by Brosch et al. (2002) and we conclude that the loss of some spacer units can be used as phylogenetic markers in a similar manner to the loss of chromosomal regions. In combination with the molecular markers described by Brosch et al. (2002) the spoligotype patterns define a series of groups of bacteria, that we call clades.
Section snippets
Spoligotypes of the RD9 deleted lineage
We have obtained the spoligotype patterns of all the strains used by Brosch et al. (2002) to define the phylogeny of the RD9 deleted lineage of the M. tuberculosis complex. The M. africanum isolates were spoligotyped at the Wadsworth Center, caprine and some bovine spoligotype patterns by S. Samper, Spain (Haddad et al., 2001) and all other patterns were obtained from the RIVM database of spoligotype patterns. A representative sample of strains from this lineage and their spoligotype patterns
The antelope-associated clade
Although the presence of certain spacers is phylogenetically informative this is not true for the absence of spacers; some spacers have been independently lost in different clades. For example, strains of M. microti from the seal/vole clade have lost all but two of the spacer units. Therefore, on its own, the absence of spacers cannot be used to define membership of a clade; it is the presence of certain spacers that is phylogenetically congruent with the deletions and SNMs used by Brosch et
Discussion
We have shown that the presence of some spoligotype spacers is phylogenetically informative for strains of the RD9 deleted lineage identified by Brosch et al. (2002) and, using the informative spoligotype spacer units, SNMs and chromosomal deletions we have defined a series of nested clades; all of the phylogenetically informative markers are congruent down this lineage. We have used the phylogenetically informative spacers as a screening method to identify further members of a group associated
Acknowledgements
We should like to thank S. Samper for supplying spoligotype patterns. This work was funded by DEFRA.
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