About Mycobacterium Tuberculosis
Mycobacterium tuberculosis is a successful worldwide human pathogen responsible for 2–3 million deaths and 8–10 million new cases per year, most of them being in resource poor countries. Genotyping of M. tuberculosis is useful for population dynamics analysis as well as the identification of outbreaks. Genotyping is based upon genomic variability in M. tuberculosis, and, using a combination of two alleles at katG463 and gyrA95, the species can be broadly divided into three major genetic groups. Fingerprinting techniques based on repetitive DNA sequences can further differentiate these groups into genetic families including the East African Indian, Beijing, Haarlem and X, and Latin American and Mediterranean families. Spoligotyping studies delineated nine major clades including genotypes responsible for major outbreaks, which were supported by a study analysing neutral variation found within genes associated with drug resistance. Deletion analysis shed further light on the deeper structure of the M. tuberculosis complex and found six main lineages and 15 sublineages of M. tuberculosis. Although the genetic markers used in these studies were different, the overall phylogenetic structure of the species was the same between the different methods and demonstrated that M. tuberculosis was clonal. Genotyping methods currently rely upon analysis of restriction profiles including pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphisms (RFLP) using IS6110 probing, amplification profiles of selected regions of variable number tandem repeat (VNTR) including the exact tandem repeat (ETR) regions and mycobacterial interspersed repetitive units (MIRU), spoligotyping and deletion and insertion site mapping. Recently, single nucleotide polymorphism (SNP) analysis including SNP located in intergenic spacers was performed, delineating either six or nine broad groups. However, systematic sequencing of intergenic spacers has not been done for M. tuberculosis genotyping.
We investigated Multispacer Sequence Typing (MST) for M. tuberculosis genotyping. This technique is based on a single sequence analysis of several intergenic regions selected by complete genome sequence comparison, resulting in a sequencing-based, genotypic profile. MST has been previously used to genotype several pathogens otherwise demonstrated to be highly homogenous, including Yersinia pestis, Bartonella quintana, Rickettsia conorii, Rickettsia prowazekii, Coxiella burnetii and Bartonella henselae. Intergenic spacers have been investigated for the identification of Mycobacterim tuberculosis complex species, but has never been applied to M. tuberculosis genotyping. We herein developed a sequencing-based approach for the genotyping of M. tuberculosis isolates from our laboratory and further compared MST with a blinded panel of 93 IS6110-RFLP and MIRU/VNTR-characterised strains.
- 2008-06-18Multispacer sequence typing for Mycobacterium tuberculosis genotypingDjelouadji Z, Arnold C, Gharbia S, Raoult D, Drancourt M