**5. The IS***6110* **content: How many are the best number?**

A high variability in number of IS*6110* is observed amongst the different strains of the MTBC. While *M. bovis* usually has a unique copy, *M. tuberculosis* varies from zero to twenty five. In any case, it is difficult to answer the question: What is the best number for the bacteria?

influence of the IS on the downstream genes is related to the distance among the gene and the 3'-end of the IS. Thus, a promoter influence is possible within the range of 31 to 300bp of

This could be due to a polar effect of the IS and also due to the presence of an outward promoter that was identified close to the 3'-end of IS*6110* (Safi et al., 2004; Soto et al.,

The promoter carried by IS*6110* has the relevance of being activated inside monocytes (Safi et al., 2004) and its activity was demonstrated in several genes not only in the strain H37Rv but also in other clinical strains including Beijing strains (Safi et al., 2004). Remarkably that promoter activity has been demonstrated by the upregulation of the main two-component

In this context, it is noteworthy the presence of the IS inserted between *dna*A-*dna*N proteins that control the genome replication. This insertion was identified in many strains including several belonging to the Beijing family (see part 4.1.3). Moreover the IS could be inserted in both directions in this region (Turcios et al., 2009; Casart et al., 2008) having thus putatively

Rv1468c (PE-PGRS)

Rv2179c (CHP\*) Rv3427c (IS*1532*)

Rv2166c, Rv2478c and Rv3188 (CHP\*) Rv2288 (FAD-cofactor containing domain)

(PPE38, PPE39 and PPE40 respectivily)

system of this bacterium, namely *pho*P/*pho*R (Soto et al., 2004).

**Reference bacteria/strain ORFs (genes)**  Soto et al., 2004 *M. bovis* Rv0757 (*pho*P/*pho*R)

Safi et al., 2004 *M. tuberculosis* Rv0797 and Rv3327 (IS*1547*)

*M. tuberculosis* Rv0002 (*dna*N)

Table 5. Identified genes located under the putative influence of the IS*6110* promoter activity. Changes in the gene expression were demonstrated in some of the cases.

Much effort should be used to complete the record of the loci in which IS*6110* was inserted. That knowledge will much help to our understanding of the mechanisms used by the

A high variability in number of IS*6110* is observed amongst the different strains of the MTBC. While *M. bovis* usually has a unique copy, *M. tuberculosis* varies from zero to twenty five. In any case, it is difficult to answer the question: What is the best number for

*M. tuberculosis*/GC1237 Rv1468c (PE-PGRS)

*M. tuberculosis* Rv2352c, Rv2353c and Rv2354c

a variable influence on the bacterial cell division.

distance among them.

2004).

Turcios et al., 2009

McEvoy et al., 2009

Alonso et al., 2011

the bacteria?

\* CHP: Conserved Hypothetical Protein.

tubercle bacilli to cause Tuberculosis so successfully.

**5. The IS***6110* **content: How many are the best number?** 

## **5.1** *M. tuberculosis* **low copy number strains (LCS) & high copy number strains (HCS)**

*M. tuberculosis* strains with less than six copies of IS*6110* are usually referred as low copy number strains (LCS) in the literature. A few clinical investigations reported the presence of LCS in regions as India, Vietnam or Tanzania. (Barlow et al., 2001; Sankar et al., 2011a). The 66% of the *M. tuberculosis* strains isolated in Tiruvallur, South India, presented a single copy of the IS*6110* or LCS (Shanmugam et al., 2011). In Kanpur district, , North India, the 17% of the *M. tuberculosis* isolates were LCS (Purwar et al., 2011). High copy number *M. tuberculosis* strains (HCS), with six or more copies of IS*6110,* were reported by a greater number of papers. One study from Brazil, reported that 93.6% of *M. tuberculosis* strains had at least six copies ranging from 1 to 18 (Suffys et al., 2000). In San Francisco, of 1,326 isolates investigated, 90% had six o more copies and only two isolates had no copies of IS*6110* (Yang et al., 1998).

A majority (96.2%) of the 183 strains fingerprinted from Kampala were HCS. These strains were isolated from patients with known HIV sero-status. The number of IS*6110* copies ranged from 1 to 20 and the frequency of occurrence of IS*6110* bands was similar between the two serogroups. The most prevalent pattern observed had 14 copies of IS*6110* with the same distribution comparing HIV seropositive and HIV seronegative patients (Asiimwe et al., 2009).

Chauhan et al analyzed 308 isolates of *M. tuberculosis* from different parts of India and 56 per cent of the isolates showed HCS of IS*6110.* At the regional level, there was not much difference in the IS*6110* copy numbers of isolates from different parts of that country (Chauhan et al., 2007).

A long term population based study analysing 1759 clinical strains from the state of Alabama showed that 65% corresponded with HCS. The results of this study demonstrated that clustering cases is clearly associated with different social factors and risk behaviors but not with high or low number of copies of the IS*6110* (Kempf et al., 2005).

### **5.2 Are there any clinical properties associated to LCS or HCS?**

After revision of the literature looking for the origin of outbreaks including MDR cases, it was evident that both LCS and HCS were involved in outbreaks at similar proportion. Some examples of large outbreaks in population studies showing different copy number strains are listed in Table 6.

The Beijing family is one of the lineages with the highest number of copies of IS*6110* (see part 4.1.3). There are controversies among the behaviour of the Beijing lineage. On the one hand, a Beijing strain named GC1237 has been responsible of epidemic outbreaks since its appeared in the community in 1993 (Caminero et al., 2001), on the other hand, one study conducted in Cape Town (South Africa) found no significant association between the *M. tuberculosis* genotype and transmissibility within the household (Marais et al., 2009). Besides, there are outbreaks reported caused by LCS, as was the extensive transmission of *M. tuberculosis* in a rural population with minimal risk factors for TB. This strain was designated as CDC 1551 and the fingerprint showed only 4 copies of the IS*6110* (Valway et al., 1998).

IS*6110* the Double-Edged Passenger 77

IS*6110* has been also checked as a tool to analyze the evolution of members of the MTBC. Transposition may have influence on the evolution of the strains, thus the parental strains should carry low copy number and the descendant, more evolved, would carry high copy number. One example that theoretically support that consideration is the Beijing family, members of this family are IS*6110* high copy-number and have shown high prevalence and high transmissibility (Mc Evoy et al., 2007). These characteristics could be seen as selective advantages of bacteria to its main purpose: infect humans (Hanekom et al., 2011). However the previous statement was theoretically possible, the presence of preferential sites, together to the presence of forbidden sites makes the study of IS*6110* variation in the genomes useless

Al-Hajoj, S.A.; Akkerman, O.; Parwati, I.; al-Gamdi, S.; Rahim, Z.; van Soolingen, D.; van

Allix, C.; Supply, P. & Fauville-Dufaux, M. (2004). Utility of fast mycobacterial interspersed

Alonso, H.; Aguilo, J.I.; Samper, S.; Caminero, J.A.; Campos-Herrero, M.I.; Gicquel, B.;

Armand S, Vanhuls P, Delcroix G, Courcol R & Lemaitre N. (2011)..Comparison of the Xpert

Asiimwe, BB.; Joloba, ML.; Ghebremichael, S.; Koivula, T.; Kateete, DP.; Katabazi, FA.;

Bannalikar AS, Verma R. (2006). Detection of *Mycobacterium avium* & *M. tuberculosis* from

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Brosch, R.; Martín, C. & Otal, I. (2011). Deciphering the role of *IS6110* in a highly transmissible *Mycobacterium tuberculosis* Beijing strain, GC1237. *Tuberculosis*

MTB/RIF test with an IS*6110*-TaqMan real-time PCR assay for direct detection of *Mycobacterium tuberculosis* in respiratory and nonrespiratory specimens. *Journal of Clinical Microbiology*. Vol.49, No.5, (May 2011), pp. 1772-1776, ISSN 0095-1137 Asgharzadeh, M.; Kafil, HS.; Roudsary, AA.; Hanifi, GR. (2011). Tuberculosis transmission

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as evolutionary tool (Kivi et al., 2002).

**6. References** 

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Because in most population-based studies the proportion of cases with isolates that have five or fewer copies of IS*6110* is low, the impact of these cases in the study of the overall transmission of tuberculosis in a community will be low.

Table 6. *M. tuberculosis* complex strains causing large outbreaks.
