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The purpose of this study was to increase our knowledge about Mycobacterium africanum and report the incidence and characteristics of tuberculosis (TB) due to their lineages in Aragon, Spain, over the period 2003–2019. The study includes all the cases in our region, where all the M. tuberculosis complex isolates are systematically characterised. We detected 31 cases of M. africanum among 2598 cases of TB in the period studied. TB caused by M. africanum is rare (1.19%) in our population, and it affects mainly men of economically productive age coming from West African countries. Among the isolates, Lineage (L) 6 was more frequent than L5. The genotyping of these strains identified five clusters and 13 strains with a unique pattern. The isolates’ characterisation identified a copy of IS6110 within the moaX gene, which turned out to be specific for L6. It will allow the differentiation of this lineage from the rest of MTBC with a simple PCR reaction. It remains to be established whether this polymorphism may limit M. africanum transmission. Furthermore, a mutation in the mutT2 promoter was found as specific for L6 strains, which could be related to the high variability found for L6 compared to L5.

f Transposition and homologous recombination of IS6110 appear in Mycobacterium tuberculosis along in vivo sequential infections. These events were checked in different clones of a successful strain, M. tuberculosis Zaragoza, with the focus on a variant in which integration of a copy of IS6110 in the origin of replication (oriC) region occurred. Clonal Mycobacterium tuberculosis variants appear along the sequential infections of hosts involved in the same transmission cluster (1). IS6110 in M. tuberculosis has been one of the most widely used elements in molecular epidemiology and has a transposition rate estimated at ϳ18% over a period of 5 to 6 years (2). The IS6110 element itself modulates expression of neighboring genes and, depending on its location, could confer both transmission ability and virulence (3-5). Microevolution events due to the transposition of IS6110 are usually reflected in variations of restriction fragment length polymorphism (RFLP) patterns (6, 7). Minor variations in IS6110 fingerprints, spoligotypes, or mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) loci reveal microevolution in clonal infections (6,8). These changes could have effects on the molecular structure of the mycobacterial genome and the biology of the bacillus (7).Molecular epidemiological studies carried out for more than a decade have provided accuracy in the study of the transmission (9). The usefulness of the approach has been proven, even in countries with low incidences of tuberculosis (10). The M. tuberculosis Zaragoza strain spread in Zaragoza, Spain, and reached 18.7% of all isolates of M. tuberculosis in 2001 to 2004 (11). This strain was classified in principal genetic group 3 and in singlenucleotide polymorphism (SNP) cluster group 6a (12), it demonstrated the rare spoligotype SIT773, and it carried 12 localized copies of IS6110 (13). In addition, in the context of a highthroughput survey of in vivo IS6110 transposition in multiple M. tuberculosis genomes, an additional IS6110 copy was detected in the oriC region in one isolate (14). The purpose of this study was to review the in vivo genomic changes of the M. tuberculosis Zaragoza strain, focusing on this variant.Among the 2,348 isolates collected between 2000 and 2013, 246 were identified as M. tuberculosis Zaragoza by IS6110 RFLP typing (15) and spoligotyping (16) in the context of tuberculosis surveillance in our region. By tracking the strain changes throughout time, we found 7 different variants, i.e., variants 1 to 7 (Fig. 1). An additional copy of IS6110 was detected in six evolved clones, namely, variants 1 to 6. Variant 7 lost one copy in the RFLP pattern and was the only variant that revealed a change in its spoligotyping pattern. Clones 1, 2, 4, 5, 6, and 7 were isolated from only one patient, whereas variant 3 was isolated from 12 patients, as described below.In variant 7, 10 of the 12 IS6110 insertion sites known for M. tuberculosis Zaragoza were confirmed by PCR (13). However, the two nonamplified copies were l...

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