Genetic diversity of Mycobacterium africanum clinical isolates based on IS6110-restriction fragment length polymorphism analysis, spoligotyping, and variable number of tandem DNA repeats

A collection of 105 clinical isolates originally identified as Mycobacteriu m africanum were characterized using both phenotypic and genotyping methods . The phenotypic methods included routine determination of cultural propert ies and biochemical tests used to discriminate among the members of the M. tuberculosis complex, whereas genotypic characterization was based on IS611 0-restriction fragment length polymorphism (IS6110-RFLP) analysis, IS1081-R FLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyp ing), variable number of tandem DNA repeats (VNTR), and the polymorphism of the oxyR, pncA, and mtp40 loci. The results obtained showed that a majorit y of M. africanum isolates were characterized by a specific spoligotyping p attern that was intermediate between those of M. tuberculosis and M. bovis, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respect ively. A tentative M. africanum-specific spoligotyping signature appeared t o be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as th e polymorphism of oxyR and pncA, a total of 24 isolates were excluded from the final study (19 were identified as M. tuberculosis, 2 were identified a s M. canetti, and 3 were identified as M. bovis). The remaining 81 M. afric anum isolates were efficiently subtyped in three distinct subtypes (A1 to A 3) by IS6110-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of t he A2 subtype but showed significant variations for subtypes A1 and A3. A p hylogenetic tree based on a selection of isolates representing the three su btypes using VNTR and spoligotyping alone or in combination confirmed the s ubtypes described as well as the heterogeneity of subtype A3.