**6. Implication of variability on disease control**

From the pool of individuals that come in contact with and are infected with *M. tuberculosis*, only about 10% will develop disease. The manifestation of the disease in these individuals, however, can vary greatly from a self-limited infection in the lungs to extra-pulmonary and disseminated cases (Nicol & Wilkinson, 2008). The outcome of infection must therefore be influenced both by host factors that may predispose to infection, and to genetic variation in the tubercle bacillus itself. Several host factors have been associated with risk for disease, such as malnutrition, vitamin D deficiency, NRAMP1 polymorphisms, diabetes and co-infection with HIV (Malik & Godfrey-Faussett, 2005). A recent study analysing the impact of pathogen variability in recombinant congenic mice indicated that host control of the infection varied depending on the infecting strain and the stage of infection. The dynamic response to disease suggests that in addition to host genetic determinants, the pathogen background also influences the outcome of infection (Di Pietrantonio *et al.*, 2010). Studies with both laboratory and clinical strains have also suggested a correlation between strain genotype and the infectious process. This correlation, however, has been difficult to resolve in great part due to the difficulty associated with working with this slow-growing pathogen and to problems associated with extrapolation from animal models (Nicol & Wilkinson, 2008). The integration of genomics and epidemiological data has been able to link some cases of genetic variability with strain phenotypic characteristics. By analysing deletions in clinical isolates it was suggested, for example, that strains causing cavitary disease had fewer deletions, indicating that the accumulation of mutations affected pathogenesis (Kato-Maeda *et al.*, 2001b). Mutations that altered the PE\_PGRS33 protein, which may be involved in cell-cell interactions and antigenic variation, have also been connected with clustering and pathogenesis and thus with clinical and epidemiological characteristics of *M. tuberculosis* isolates (Talarico *et al.*, 2007). Similarly, an analysis of the genetic variation at the *plcD* locus indicated that variability in this region was possibly associated with pathogenesis and disease manifestation (Yang *et al.*, 2005). Studies involving strains that cause pulmonary and extra-pulmonary infections have also indicated that extra-respiratory strains were more efficient at infecting macrophages and could also have higher infectivity *in vivo* (Garcia de Viedma et al., 2005).

The growing consensus that the main MTBC lineages are associated with geographic origin suggests co-evolution of lineages with their hosts and thus adaptation that must involve events of strain variation. More recent evidence of the restricted geographical niche of certain lineages came from an Ibero-America MDR *M. tuberculosis* survey showing that circulation of Latin American MDR strains was restricted to particular areas and also that transnational transmission was scarce (Ritacco *et al.*, 2011). The Beijing lineage, one of the most extensively studied families, has been responsible for several epidemic outbreaks and

of copies of the IS*6110* are also epidemiologically successful. In general, though, there is still insufficient information regarding the factors that influence the frequency of transposition, such as the genomic context of the insertion element within a particular strain background. The variation in the number of IS*6110* elements among *M. tuberculosis* isolates also raises the possibility that copy number is the result of the evolution of particular lineages as strains cope with IS*6110* transposition and its resulting genetic variability, and in some cases even selecting for phenotypically favorable events, while keeping genome integrity and avoiding

From the pool of individuals that come in contact with and are infected with *M. tuberculosis*, only about 10% will develop disease. The manifestation of the disease in these individuals, however, can vary greatly from a self-limited infection in the lungs to extra-pulmonary and disseminated cases (Nicol & Wilkinson, 2008). The outcome of infection must therefore be influenced both by host factors that may predispose to infection, and to genetic variation in the tubercle bacillus itself. Several host factors have been associated with risk for disease, such as malnutrition, vitamin D deficiency, NRAMP1 polymorphisms, diabetes and co-infection with HIV (Malik & Godfrey-Faussett, 2005). A recent study analysing the impact of pathogen variability in recombinant congenic mice indicated that host control of the infection varied depending on the infecting strain and the stage of infection. The dynamic response to disease suggests that in addition to host genetic determinants, the pathogen background also influences the outcome of infection (Di Pietrantonio *et al.*, 2010). Studies with both laboratory and clinical strains have also suggested a correlation between strain genotype and the infectious process. This correlation, however, has been difficult to resolve in great part due to the difficulty associated with working with this slow-growing pathogen and to problems associated with extrapolation from animal models (Nicol & Wilkinson, 2008). The integration of genomics and epidemiological data has been able to link some cases of genetic variability with strain phenotypic characteristics. By analysing deletions in clinical isolates it was suggested, for example, that strains causing cavitary disease had fewer deletions, indicating that the accumulation of mutations affected pathogenesis (Kato-Maeda *et al.*, 2001b). Mutations that altered the PE\_PGRS33 protein, which may be involved in cell-cell interactions and antigenic variation, have also been connected with clustering and pathogenesis and thus with clinical and epidemiological characteristics of *M. tuberculosis* isolates (Talarico *et al.*, 2007). Similarly, an analysis of the genetic variation at the *plcD* locus indicated that variability in this region was possibly associated with pathogenesis and disease manifestation (Yang *et al.*, 2005). Studies involving strains that cause pulmonary and extra-pulmonary infections have also indicated that extra-respiratory strains were more efficient at infecting macrophages and could

deleterious effects.

**6. Implication of variability on disease control** 

also have higher infectivity *in vivo* (Garcia de Viedma et al., 2005).

The growing consensus that the main MTBC lineages are associated with geographic origin suggests co-evolution of lineages with their hosts and thus adaptation that must involve events of strain variation. More recent evidence of the restricted geographical niche of certain lineages came from an Ibero-America MDR *M. tuberculosis* survey showing that circulation of Latin American MDR strains was restricted to particular areas and also that transnational transmission was scarce (Ritacco *et al.*, 2011). The Beijing lineage, one of the most extensively studied families, has been responsible for several epidemic outbreaks and in some cases has been associated with multidrug resistance (Hanekom *et al.*, 2011). Its capacity to spread within a population is evident from epidemiological studies and emphasizes the possibility that certain strain properties could contribute to this lineage's expansion in the population (Nicol & Wilkinson, 2008). The increased virulence of these isolates was associated with the production of a phenolic glycolipid (PGL) that affects the host immune response, and which is absent in many other *M. tuberculosis* families (Ordway *et al.*, 2007, Hanekom et al., 2011). More recent work suggests that although PGL can contribute to *M. tuberculosis* virulence, it probably requires additional bacterial factors (Sinsimer *et al.*, 2008). Other examples stem from studies of strains that have caused outbreaks, such as strains CDC1551 and HN878, the latter also a member of the Beijing family. In these and other studied cases, it appears that some of the effects observed have to do with the capacity of these strains to induce variable inflammatory responses (Coscolla & Gagneux, 2010). Despite these studies, many of the clinical outcomes associated with strain variability still need to be further examined, particularly in other *M. tuberculosis* lineages before precise genotypic variability can be associated with phenotypic differences.

The emergence and spread of drug-resistant strains is particularly disturbing and provides additional examples where strain variability can have a profound effect on disease outcome. One particularly alarming case was the epidemic caused by an XDR strain in the KwaZulu-Natal region of South Africa that resulted in high mortality, causing the death of 52 of the 53 patients co-infected with HIV in the course of 16 days (Gandhi *et al.*, 2006). To understand more about the dynamics of appearance and dispersion of this highly virulent KZN strain, whole genome sequence analysis was carried out for XDR, MDR and drug sensitive KZN strains. The results indicated that the outbreak was most probably due to clonal expansion of a single strain and that a particular strain genetic background did not necessarily contribute to acquisition of antibiotic resistance (Ioerger *et al.*, 2009). Further work will be needed to better understand this strain's virulence and transmissibility in the community.

Part of the success of *M. tuberculosis* as a human pathogen is due to its capacity to be efficiently transmitted between hosts and to persist for long periods of time despite the host's immune response. A recent study involving whole genome sequencing of 21 strains from the six main *M. tuberculosis* lineages indicated that human T cell epitopes had very little sequence variation and were highly conserved relative to the rest of the genome. It was suggested that these antigens, contrary to expectations, might be under purifying selection and be benefitting from host immune recognition (Comas *et al.*, 2010). This differs from the classical view of immune evasion due to the selective pressure imposed by the immune response and may indicate that new approaches should be considered for vaccine development and control of *M. tuberculosis*.

The genetic variability evident in strains of the MTBC bears relevance to control of tuberculosis since treatment must work against all circulating strains. Rapid and accessible diagnostics for both *M. tuberculosis* and drug resistant isolates are still required, as is the availability of a vaccine that can be universally effective, given the variable efficacy of the currently used BCG vaccine. There are now more that 10 vaccines under phase I trial and the hope is that in the near future at least one of these will prove to be safe and protective by containing *M. tuberculosis* and preventing reactivation. However, future strategies will need to address the need to prevent or eradicate latent infections, especially in view of additional factors affecting disease and the host immune response, such as co-infection with HIV

Genomic Variability of *Mycobacterium tuberculosis* 51

Work Programme of the 7th Framework Programme (G.A. no. 200999)

Abadia, E., J. Zhang, T. dos Vultos, V. Ritacco, K. Kremer, E. Aktas, T. Matsumoto, G.

Alexander, K. A., P. N. Laver, A. L. Michel, M. Williams, P. D. van Helden, R. M. Warren &

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

Alland, D., D. W. Lacher, M. H. Hazbon, A. S. Motiwala, W. Qi, R. D. Fleischmann & T. S.

Becq, J., M. C. Gutierrez, V. Rosas-Magallanes, J. Rauzier, B. Gicquel, O. Neyrolles & P.

Borrell, S. & S. Gagneux, (2011) Strain diversity, epistasis and the evolution of drug resistance in *Mycobacterium tuberculosis*. *Clin Microbiol Infect* 17: 815-820. Brosch, R., S. V. Gordon, M. Marmiesse, P. Brodin, C. Buchrieser, K. Eiglmeier, T. Garnier, C.

Brosch, R., S. V. Gordon, A. Pym, K. Eiglmeier, T. Garnier & S. T. Cole, (2000) Comparative

Brosch, R., A. S. Pym, S. V. Gordon & S. T. Cole, (2001) The evolution of mycobacterial pathogenicity: clues from comparative genomics. *Trends Microbiol* 9: 452-458. Brudey, K., I. Filliol, S. Ferdinand, V. Guernier, P. Duval, B. Maubert, C. Sola & N. Rastogi,

Caws, M., G. Thwaites, S. Dunstan, T. R. Hawn, N. T. Lan, N. T. Thuong, K. Stepniewska, M.

utility of LSPs in phylogenetic analysis. *J Clin Microbiol* 45: 39-46.

evolution of the tubercle bacilli. *Mol Biol Evol* 24: 1861-1871.

*tuberculosis* complex. *Proc Natl Acad Sci U S A* 99: 3684-3689.

genomics of the mycobacteria. *Int J Med Microbiol* 290: 143-152.

Refregier, D. van Soolingen, B. Gicquel & C. Sola, (2010) Resolving lineage assignation on *Mycobacterium tuberculosis* clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method. *Infect Genet* 

N. C. Gey van Pittius, (2010) Novel *Mycobacterium tuberculosis* complex pathogen,

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

Whittam, (2007) Role of large sequence polymorphisms (LSPs) in generating genomic diversity among clinical isolates of *Mycobacterium tuberculosis* and the

Deschavanne, (2007) Contribution of horizontally acquired genomic islands to the

Gutierrez, G. Hewinson, K. Kremer, L. M. Parsons, A. S. Pym, S. Samper, D. van Soolingen & S. T. Cole, (2002) A new evolutionary scenario for the *Mycobacterium* 

(2006) Long-term population-based genotyping study of *Mycobacterium tuberculosis* complex isolates in the French departments of the Americas. *J Clin Microbiol* 44:

N. Huyen, N. D. Bang, T. H. Loc, S. Gagneux, D. van Soolingen, K. Kremer, M. van der Sande, P. Small, P. T. Anh, N. T. Chinh, H. T. Quy, N. T. Duyen, D. Q. Tho, N. T. Hieu, E. Torok, T. T. Hien, N. H. Dung, N. T. Nhu, P. M. Duy, N. van Vinh Chau & J. Farrar, (2008) The influence of host and bacterial genotype on the development of disseminated disease with *Mycobacterium tuberculosis*. *PLoS Pathog* 4: e1000034 Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K.

Eiglmeier, S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin,

(http://cordis.europa.eu/fp7/dc/index.cfm).

M. mungi. *Emerg Infect Dis* 16: 1296-1299.

*Evol* 10: 1066-1074.

91: 117-126.

183-191.

**9. References** 

(Kaufmann, 2010). New and alternative drugs are also required to shorten the current duration of chemotherapy, to act against persistent bacilli and to counteract the spread of drug-resistant strains that frustrate global eradication programs. Due to renewed efforts in recent years, several novel drugs have been identified and are under clinical evaluation or being developed, many of which involve novel targets and mechanisms (Coxon & Dover, 2011). The discovery of novel drugs has involved different approaches that include the use of genomics to identify targets, whole-cell screening and re-engineering of known chemical molecules (Koul *et al.*, 2011). Given the observed strain variability it is nonetheless possible that some of these drugs might vary in efficiency in different strain backgrounds, as was made evident for DGC and Cyp121 in the Haarlem lineage (Cubillos-Ruiz et al., 2010). Thus the heterogeneity among different strains and lineages, as well as of the host-pathogen interaction, must be taken into account when developing novel diagnostics and therapeutic strategies. Extensive analysis of circulating *M. tuberculosis* populations will be required to address the efficacy of treatment and vaccination in different genetic backgrounds. The advent of novel massive sequencing techniques to generate genomic data for multiple strains will undoubtedly allow examination of whole genomes and make such analyses more feasible (Lin & Ottenhoff, 2008).
