**4. The tails: Risks of IS***6110*

Understanding the changes that occur in genomes among isolates of *M. tuberculosis* would give insights into their corresponding differences causing disease.

Many mechanisms can be related to changes in the bacterial genomes, being those mediated by ISs one of the most relevant and better studied (Galas & Chandler, 1989). According to general data, it was considered that among 5 to 15% of spontaneous mutations in the bacterial genomes were due to changes in the ISs locations.

The more common mechanism used by IS to move along genomes is transposition following the enzymatic activity of their encoded transposases, this transposition could lead to the generation of 3-4bp direct repeats (DR) immediately flanking the IS sequences, as it occurs to IS*6110* (Thierry et al., 1990b). Recombination is also another mechanism participating in the changes of the location of ISs along the genomes. All those mechanisms lead to IS mediated gene rearrangements, inversions, deletions etc in the bacterial genomes.

ISs could have also some polar effect on the flanking genes, particularly on downstream genes. It has been demonstrated the occurrence of gene activation due to the presence of out-warding promoters within the elements as well as the formation of new promoters upon insertion (Galas & Chandler, 1989).

different patients present the same genotype, transmission may have occurred between them. However, once transmission has occurred, the genotypes may change, resulting in divergent fingerprints. The advantage of IS*6110* as marker is that the clock of change of the IS*6110* patterns was determined in serial isolates; the half-life was extrapolated to be 3.2 years. These changes were predicted more common for persons with extrapulmonary disease and for those who had both pulmonary and extrapulmonary isolates. This fact supported the use of IS*6110* typing in epidemiologic studies of recent transmission of TB (de Boer et al., 1999). The results of a study carried out to estimate the recent transmission based on IS*6110*-RFLP suggested that the interpretation of the recent transmission index, and the resulting necessary public health interventions, will vary according to how researchers account for spontaneous mutation when estimating transmission from the genotyping data

In spite of all the studies carried out with this genomic element, some limitations have been found. Besides the technical difficulties that IS*6110* typing presents for some laboratories (the long time that the mycobacteria requires to growth, the equipment and the software required for the analysis), this method have also demonstrated difficulties for differentiating LCS*,* including *M. bovis* strains and is unable to identify strains with cero copies. Some studies have solved this problem by applying a second technique for these cases (Thong-On et al., 2010). Other studies with high prevalence of strains with LCS do not recommend this technique in their settings (Asgharzadeh et al., 2011). Mixed infections represent another limitation, which could be underestimated using IS*6110*-RFLP and could be confused with exogenous reinfection (Shamputa et al., 2006). The mixed tuberculosis infection suspected as a result of the IS*6110-*RFLP method could be clearly identified by MIRU-VNTR typing, which is more sensitive for the detection of multiple *M. tuberculosis* strains (Allix et al., 2004).

Understanding the changes that occur in genomes among isolates of *M. tuberculosis* would

Many mechanisms can be related to changes in the bacterial genomes, being those mediated by ISs one of the most relevant and better studied (Galas & Chandler, 1989). According to general data, it was considered that among 5 to 15% of spontaneous mutations in the

The more common mechanism used by IS to move along genomes is transposition following the enzymatic activity of their encoded transposases, this transposition could lead to the generation of 3-4bp direct repeats (DR) immediately flanking the IS sequences, as it occurs to IS*6110* (Thierry et al., 1990b). Recombination is also another mechanism participating in the changes of the location of ISs along the genomes. All those mechanisms lead to IS

ISs could have also some polar effect on the flanking genes, particularly on downstream genes. It has been demonstrated the occurrence of gene activation due to the presence of out-warding promoters within the elements as well as the formation of new promoters upon

mediated gene rearrangements, inversions, deletions etc in the bacterial genomes.

give insights into their corresponding differences causing disease.

bacterial genomes were due to changes in the ISs locations.

(Benedetti et al., 2010).

**4. The tails: Risks of IS***6110*

insertion (Galas & Chandler, 1989).

All those changes could be a risky to the bacteria's genomes integrity, being the carriage of mobile IS either a potential enemy with deadly influence on the bacterial fitness or a helpful ally contributing to the improvement of that fitness. Our current knowledge on how the IS*6110*-mediated mutations influence in the genome plasticity of the *M. tuberculosis* genome will be reviewed herewith.

## **4.1 Moving along the genome**

The numerous studies published on IS*6110*-RFLP with epidemiological purposes showed a high level of variability in the locations of this IS along the *M. tuberculosis* genome (see part 3.2). On the basis of those results the rate of transposition of IS*6110* was estimated to be about 18% over a period of 5-6 years. However it seems evident that the events of transposition are related to changes in the environment in which the bacteria are involved. It was suggested that transpositional events occur following mutational burst instead of following a constant mutation rate; this can explain the observation that changes in RFLP patterns would occurred more frequently during transmission and before diagnosis (soon after the bacilli enter inside the host) or after relapses or any other main event during the course of the infection (Schürch et al., 2010). In agreement to this consideration, two rather different half-life times were calculated for the IS*6110*-RFLP patterns stability in serial patient's isolates: 0.6 and 10.7 years; this most probably be due to changes in the patient's management or to the course of the infection in the different settings compared (Schürch et al., 2010).

Independently of why, how or when its transpositions occurred, IS*6110* mediates genome plasticity of members of the MTBC, and that plasticity is ongoing both under controlled environment *in vitro* and during infection *in vivo* (Fang et al., 1999b).

To confirm the last assertion, some papers described changes in the RFLP pattern during infection. This is showing that microevolution of the bacilli mediated by IS could occur not only during transmission between patients but also during the course of the disease in a single patient (Al-Hajoj et al., 2010). Besides, the comparison of the whole-genomes of six different H37Rv strains, collected from several laboratories, showed that multiple IS*6110* transposition events have occurred in the genome even under *in vitro* "controlled" environments (Ioerger et al., 2010).
