**1.1 Persistence of the pathogen is the hallmark of TB pathogenesis**

Based on a randomized clinical trial conducted by British Medical Council between 1972 and 1974, the World Health Organization (WHO) and other government agencies implemented a short-course multi-drug regimen for tuberculosis – a disease caused by the infection of *Mycobacterium tuberculosis (BMC, 1972, Fox et al., 1999)*. The regimen is made of three antibiotics, isoniazid, rifampicin and pyrazinamide administered over a period of six months. The extended therapy is essential for sterilizing a small subpopulation of bacilli that presumably acquire phenotypic tolerance to antibiotics (Saltini, 2006, Jindani et al., 2003).

Four decades later, WHO estimates that about 2 billion people in the world still remain asymptomatically infected with *M. tuberculosis*, approximately 5-10% of these visit clinics with symptoms of active tuberculosis, and 1.7 million die of the infection every year (Dye *et al.*, 2009). Moreover, one third of the mortality in HIV-infected patient occurs due to coinfection of *M. tuberculosis*, often with a very high frequency of multi-drug resistant strains (Harrington, 2010, Aaron *et al.*, 2004). It is thus clear that while the existing anti-TB drug regimen has been able to reduce the mortality rate, it has been inadequate in reducing the global burden of the disease. A forward approach towards TB-control must include two critical capabilities: a) to predict and prevent the conversion of asymptomatic infection to active TB, and b) to develop a shorter and more effective therapeutic regimen for active disease. Accomplishing these goals have been difficult because of our limited understanding of the mechanisms employed by *M. tuberculosis* to persist against the challenges of competent host immune system and antibiotics.

Although persistence mechanisms of *M. tuberculosis* in the host remain largely unclear, persistence of most, if not all, microbial species is facilitated by growth and existence in surface-associated and organized communities – called biofilms (Costerton *et al.*, 1999, Fux *et al.*, 2005, Hall-Stoodley *et al.*, 2004, Blankenship & Mitchell, 2006, Branda *et al.*, 2005). Several mycobacterial species including *M. tuberculosis* are now known to spontaneously grow *in vitro* as biofilms that harbor drug tolerant bacilli. This raises questions as to whether biofilms

Biofilms of *Mycobacterium tuberculosis*: New Perspectives of an Old Pathogen 183

Despite the demonstration of a non-replicative and physiologically tolerant state of *M. tuberculosis in vitro* as well as the presence of hypoxic environment in granulomas (Via *et al.*, 2008), the hypothesis that the persisters in latent infection and chemotherapy are exclusively the non-replicating subpopulation residing in the bacteriostatic condition of closed lesions remains untested (Gomez & McKinney, 2004, Parrish *et al.*, 1998). In contrast, the notion of a non-replicative state of persisters during latency is strongly challenged by two interesting studies published recently. Using an unstable plasmid as a reporter, Sherman and colleagues found that *M. tuberculosis* bacilli actively replicate during the chronic phase of infection in a mouse model – a phase when neither the host develops any symptoms of disease nor the number of live bacteria changes (Gill *et al.*, 2009). Recently, Fortune and colleagues determined that mutations in *M. tuberculosis* populations accumulate at the same rate in latent and active infections of non-human primates, and both were similar to a logarithmically growing *in vitro* culture, implying active DNA replication and thus cell

The replicative state of the bacilli in asymptomatic infection of animal models reflects a dynamic host-pathogen interface. This interestingly is fully consistent with an emerging picture of a spectrum of disease status- in terms of bacterial load, inflammation and lesion morphologies – as against the dogmatic view of a bimodal existence of infection in either latent or active form (DB *et al.*, 2009, Rhoades *et al.*, 2005, Barry *et al.*, 2009). Interestingly, comparative studies of latent and active TB not only fail to establish a clear immunological distinction but also reveal highly heterogeneous lesion morphologies reflecting localized and highly diverse host pathogen interactions within an infected organ irrespective of the clinical symptoms (Barry et al., 2009). It is thus reasonably evident that in an asymptomatic infection *M. tuberculosis* could persist in diverse physiological states – from non-replicative to fully replicative states – each with distinct host-pathogen interactions. Furthermore, persistence of actively growing bacilli in asymptomatic infection could conceivably occur through delicately balanced host-pathogen interaction, which keeps the inflammation below the symptomatic threshold, but has the greatest chance of tipping the balance to cause the

Mechanisms of persistence of *M. tuberculosis* during chemotherapy, like latency, also remains unclear, but data from clinical trials indicate a strong positive correlation between bacterial burden and duration of chemotherapy [reviewed in (Connolly *et al.*, 2007)]. Consistent with these data, the Center for Disease Control of the United States recommends an extension of chemotherapy from six to nine months in case of patients with cavitary TB (CDC, 2003). Besides the total burden, the most intriguing aspect of long-term chemotherapy in TB is that the clearance of the pathogen follows a biphasic pattern as clearly demonstrated by Mitchison and colleagues (Jindani et al., 2003) (Fig. 1). While > 95% of the population could be cleared in the first few days of the beginning of treatment, the

In summary, the persistence of *M. tuberculosis* in a chronic infection and chemotherapy are likely to be facilitated by multiple mechanisms including the adaptive changes in the bacilli in response to dynamic microenvironments during colonization and active growth. These

remaining fraction required a prolonged exposure (Jindani et al., 2003).

**3. Changing paradigms of** *M. tuberculosis* **persistence** 

division of the pathogen in latent infection (Ford *et al.*).

active disease.

could also be an *in vivo* persistence mechanism of *M. tuberculosis*. In this chapter we will discuss why it is reasonable to pay serious heed to this question, and what approaches can be used to test this hypothesis.
