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MultiLocus Sequence Typing (MLST) (Maiden et al. 1998) is a tool that is widely used for phylogenetic typing of bacteria. MLST follows the same principle as MultiLocus Enzyme Electrophoresis (MLEE), but uses DNA sequencing instead of protein electrophoresis. MLST is based on PCR amplification and sequencing of internal fragments of a number (usually 6 or 7) of essential or housekeeping genes spread around the bacterial chromosome. The differences between the sequences are used to define allelic profiles, or sequence types (STs). The genetic relatedness among isolates is then determined by comparison of the sequences or STs (see figure below). MLST is thus a method that is unambiguous and truly portable electronically among laboratories, for these reasons MLST has recently supplanted MLEE. Since the initial development of this technique for Neisseria meningitidis in 1998, MLST schemes have been developed for the most important bacterial pathogens, including Streptococcus pneumoniæ, Streptococcus pyogenes, Hæmophilus influenzæ, Staphylococcus aureus, Campylobacter jejuni, Enterococcus fæcium, Escherichia coli, and Salmonella, and schemes are being developed for many other species (see Maiden 2006 for a recent review). These MLST schemes have been used successfully to explore the population structure of bacteria, to study the evolution of their virulence properties, and to identify antibiotic−resistant strains and epidemic clones.
Our research group, led by Prof. Anne-Brit Kolstø at the Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, has designed and applied the first MLST scheme for phylogenetic analysis of B. cereus group bacteria (Helgason et al. 2004), leading to the identification of several clonal lineages comprising strains isolated from clinical sources. Alternative schemes have been subsequently developed by several other groups (Ko et al. 2004; Priest et al. 2004; Candelon et al. 2004; Sorokin et al. 2006). Web-based databases for the Priest et al. 2004 and the Sorokin et al. 2006 schemes are available at http://pubmlst.org/bcereus/ and http://spock.jouy.inra.fr/cgi-bin/bacilliMLSopen.cgi, respectively. Since 2004 MLST has been extensively used as the main typing method for analyzing the genetic relationships within the whole B. cereus group population (see the MLST Schemes page of the SuperCAT database for a list of B. cereus group MLST publications).
However, all B. cereus group MLST schemes are based on different gene and isolate sets, which makes results difficult to compare. Therefore, we have designed a combined and optimized scheme based on 3 genes from the Helgason et al. 2004 scheme, 3 genes from the Priest et al. 2004 scheme, and one gene from the Sorokin et al. 2006 scheme. This new scheme is described in Tourasse, Helgason et al. 2006. To date, 239 B. cereus group isolates have been analyzed using the combined MLST scheme. Data are available in the Tourasse-Helgason database.
Furthermore, in order to provide the B. cereus group research community with a common MLST resource and means for building a comprehensive genetic analysis of the group, we have developed a new integrated MLST database, SuperCAT, that compiles all MLST data from the 5 published schemes for the B. cereus group (Tourasse and Kolstø 2008). We used supertree techniques to combine the phylogenetic information from analysis of all MLST schemes and datasets, in order to produce an integrated view of the B. cereus group population. In particular, the strains with complete genome sequences (currently 92), for which all MLST loci are thus available, can be used to join the schemes by supertree analysis. The current dataset contains a total of 1514 isolates that have been typed by some or all of 26 gene fragments from 25 different genes.
We have recently extended the supertree approach to integrate MLST data with phylogenetic information from MultiLocus Enzyme Electrophoresis (MLEE) and Amplified Fragment Length Polymorphism (AFLP), two other typing methods that have been used for large-scale population studies of the B. cereus group. The combined MLST, MLEE, and AFLP data have been incorporated into the new HyperCAT database containing data for 3193 isolates.
Below is an overview of the MLST procedure (for details see Maiden et al. 1998 and Maiden 2006):
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