Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping

Background: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. Methods and Findings: In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing M<8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts <= 42 cycles in internal transcribed spacer-real time PCR. Conclusions: These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst)

DNA polymorphism in Mycobacterium paratuberculosis, "wood pigeon mycobacteria," and related mycobacteria analyzed by field inversion gel electrophoresis

Mycobacterium paratuberculosis strains, mycobacteria from patients suffering from Crohn's disease, "wood pigeon mycobacteria," and representatives of Mycobacterium avium-Mycobacterium intracellulare were compared by restriction endonuclease DraI digestion and field inversion gel electrophoresis. Characteristic profiles were seen for M. paratuberculosis, including isolates from patients suffering from Crohn's disease, for wood pigeon mycobacteria, and for M. avium-M. intracellulare serotypes 2, 16, 18, and 19. Two M. paratuberculosis strains used for vaccine production (St 18 and 316 F) presented patterns different from those of the other M. paratuberculosis strains. Strains St 18 yielded a pattern identical to that of the M. avium type strain serotype 2, whereas 316 F gave a unique pattern. The method developed in this study represents a useful taxonomic tool for the identification and classification of mycobacteria.</jats:p

Use of different molecular typing techniques for bacteriological follow-up in a clinical trial with AIDS patients with Mycobacterium avium bacteremia

One hundred ninety-six Mycobacterium avium isolates from blood samples recovered from 93 AIDS patients for several months were typed by serotyping, by IS1245 restriction fragment length polymorphism (RFLP) analysis and in some cases RFLP analysis with plasmids pVT2 and pLR7 as probes, and by pulsed-field gel electrophoresis (PFGE). PCR typing of single colonies was also used to detect polyclonal infections. Strains belonged mainly to serotypes 1, 4, and 8. pVT2- and pLR7-related plasmids were detected in strains from 49% of the patients. The IS1245 RFLP and PFGE analyses showed a 96.8% diversity of the M. avium strains from the 93 patients. The vast majority (95.2%) of infections were monoclonal, indicating that recent infection is unlikely, even at an advanced stage of AIDS. For one patient, sequential isolates gave divergent patterns of sensitivity and resistance to clarithromycin, but all were identified as the initial clone. RFLP analysis and PCR typing of single colonies allowed for the detection of three polyclonal infections during the bacteriological follow-up. Among strains from patients whose samples were positive by culture after treatment for 2 to 15 months, 97.4% were the same as the initial strain. In conclusion, relapses and failures were mostly due to the initial strain. These relapses and failures resulted either from the selection of resistant mutants or the reappearance of sensitive strains, suggesting the persistence of nonsterilized tissue reservoirs.</jats:p

Rapid discrimination of Mycobacterium tuberculosis complex strains by ligation-mediated PCR fingerprint analysis

A ligation-mediated PCR (LMPCR) method for the amplification of sequences flanking the IS6110 of the Mycobacterium tuberculosis complex has been developed. The method uses one primer specific for IS6110 and a second specific for a linker ligated to SalI-restricted genomic DNA. LMPCR is a rapid screening method, valuable for the fingerprinting of M. tuberculosis complex strains.</jats:p