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

182 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

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

Early glimpses of the unique adaptability of *M. tuberculosis* appeared in two landmark studies conducted in the early 20th Century. First, Corper and Cohn observed that 24 out of 56 *in vitro* cultures of human and bovine isolates contained culturable tubercle bacilli even after 12 years of incubation in sealed containers (Corper & Cohn, 1933). This *in vitro* study revealed the characteristic persistence of *M. tuberculosis* in bacteriostatic condition. Concurrent with this *in vitro* study, Opie and Aronson reported the presence of virulent *M. tuberculosis* bacilli in about 26% of lesions resected from individuals dying of causes unrelated to TB (Opie & Aronson, 1927). While this study demonstrated asymptomatic infection of *M. tuberculosis*, it also opened up questions as to how bacilli are able to evade the immune system and suppress inflammation. In subsequent follow-up studies it appeared that the bacilli were unexpectedly present in uninvolved tissues instead of the presumed primary lesions (Feldman & Baggenstoss, 1939). These *in vivo* studies thus raised speculations that a competent immune system is capable of clearing the bacilli at the primary lesions, but the bacilli could have used escape mechanism to survive at secondary

The persistent nature of *M. tuberculosis* re-occupied the spotlight of tuberculosis research during the early phase of antibiotic-era around mid-20th Century. In several bacteriological studies on resected lesions from antibiotic treated individuals the bacilli could be microscopically observed even though individuals had converted to sputum negative (Loring & Vandiviere, 1956, Loring *et al.*, 1955, Vandiviere *et al.*, 1956, 1953). Interestingly, these bacilli in many instances were non-culturable and often associated with resolved lesions, thus raising a debate whether these were dead, or viable but non-culturable bacilli. The idea of viable but non-culturable bacilli seemed more convincing after McDermott and colleagues demonstrated reactivation of TB in mice upon termination of chemotherapy that was sufficient to reduce viability to undetectable levels (McCune *et al.*, 1966). It was, however, not clear in this study as to how and where the viable bacilli persisted, but the correlation between non-culturable bacilli and closed hypoxic lesions (Vandiviere et al., 1956, Haapanen *et al.*, 1959) support the idea that closed lesions could possibly be the primary site of non-replicating persisters which developed in the bacteriostatic environment

Several attempts have been made to investigate the physiology of non-replicating persisters using *in vitro* models such as hypoxic and nutritionally starved cultures. These studies subsequently led to identification of genetic components responsive to these conditionsmost notably *isocitrate lyase* (*icl*) of the glyoxalate shunt pathway and the two-component regulatory system *dosR-dosS (Park et al., 2003, Hobby & Lenert, 1957, Wayne & Hayes, 1996, Wayne & Lin, 1982, Saini et al., 2004)*. While the mutation in *icl* impairs the persistence of bacilli in a mice model (McKinney *et al.*, 2000), the phenotype of *dosR-dosS* mutants in animal models have yielded conflicting results (Rustad *et al.*, 2008, Parish *et al.*, 2003,

**2. A historical perspective of studies on** *M. tuberculosis* **persistence** 

be used to test this hypothesis.

sites in presumably a non-replicating state.

of the lesions.

Malhotra *et al.*, 2004).

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

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 active disease.

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

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

Biofilms of *Mycobacterium tuberculosis*: New Perspectives of an Old Pathogen 185

Chu *et al.*, 2006, Blankenship & Mitchell, 2006, Branda et al., 2005, Danese *et al.*, 2000, Higgins *et al.*, 2007). The constituent microbes in biofilms must reside in, and therefore adapt to, highly complex, heterogeneous and dynamic microenvironments that conceivably could foster phenotypic diversity in the population, a scenario unlikely to be encountered by single-cell planktonic counterparts (Kolter & Losick, 1998). Overall, the encapsulated growth along with phenotypic diversity in the population can be argued as the primary contributors to the extraordinary persistence of biofilms against environmental challenges including

The changes in intercellular interactions, cellular physiology and structural compositions associated with development of pathogenic biofilms can also have a profound effect on the outcome of both acute and chronic infections. Accumulation of a set of two quorum sensing signals, CAI-1 and AI-2, in high density cultures of *Vibrio cholerae* negatively co-regulate genes for ECM synthesis as well as virulence (Higgins et al., 2007). This suggests that formation of biofilms and creation of suitable microenvironments in the host through virulence factors are intricately related steps that constitute the colonization phase of an acute infection of *V. cholerae*, and their concomitant down-regulation at high density could possibly be an exit strategy of the pathogen. However, in a chronic infection of *S. aureus* in a mouse model Shirtliff and colleagues found that early and late stages of biofilms elicit distinct host responses (Prabhakara *et al.*, 2011). While early stage biofilms triggered a Th1 mediated acute inflammatory response- possibly to create conducive tissue microenvironment for colonization – the old biofilms induced Th2-mediated humoral response that was ineffective on the pathogen – perhaps an immune evasive mechanism

Taken together, biofilms represent a natural but highly complex life-style of most microbial species, promote persistence of constituent cells in robust structures, and provide unique

While the long-term persistence of *M. tuberculosis* against the host immune system and antibiotics has striking similarity with the chronic infections of biofilm forming pathogens, it remains unclear if the tubercle bacilli form biofilms in the host. It is, however, noteworthy that *in vitro* cultures of all mycobacterial species grow in complex structures that eventually develop as pellicles on the liquid-air interface, unless a detergent is added as dispersal agent in the medium. Interestingly, such growth pattern of mycobacteria have frequently been noted in the literature as aggregation of cells driven by their surface hydrophobicity, and largely been ignored ever since Dubos and colleagues reported a method to grow dispersed culture of tubercle bacilli without diminishing their virulence (Dubos *et al.*, 1946). However, the emerging concept of microbial persistence in biofilms have recently led several groups to investigate the detergent-free *in vitro* growth of mycobacterial species from the perspective of organized multicellular structures (Hall-Stoodley & Lappin-Scott, 1998, Carter *et al.*, 2003). In one of the first genetic studies of surface associated growth of mycobacteria, Kolter and colleagues observed that an *M. smemgatis* mutant deficient in biosynthesis of acetylated glycopeptidolipid was also unable to attach and grow on an abiotic surface, thus demonstrating a specific genetic requirement for surface-associate mycobacterial growth (Recht & Kolter, 2001). Ojha *et al.* subsequently reported that a

antibiotics (Mah & O'Toole, 2001).

that facilitates the chronic survival (Prabhakara et al., 2011).

microenvironments that facilitate extensive phenotypic diversity.

**5. Could** *M. tuberculosis* **infections persist as biofilms?** 

changes could either be in surface structure or physiology that lead to decreased antibiotic permeability, as well as controlled host-pathogen interaction and inflammation. Therefore, addressing questions such as where and how *M. tuberculosis* colonizes during chronic infection and gaining insight into the growth phase-dependent adaptive changes are critical for a comprehensive understanding of its persistence.

Fig. 1. Representation of the data published by Jindani et al. (4), showing the pattern of *M. tuberculosis* clearance in patients treated with isoniazid and rifampicin.
