**4. Chronic infections, bacterial persistence and biofilms**

The hallmark of a successful pathogen is to colonize the host for a long period of time against the challenges of host immune system, and persist against the antibiotics pressure. Although numerous bacterial and fungal pathogens including *M. tuberculosis* easily qualify for this category, the question as to how they establish such infections remains unanswered for most species.

However, a large and ever-growing body of evidence provide a compelling argument that the persistence of most, if not all, microbial species in general is achieved through their ability to grow in self-organized, surface associated, sessile communities called biofilms (Costerton et al., 1999, Fux et al., 2005, Hall-Stoodley et al., 2004, Kolter & Greenberg, 2006, Marrie *et al.*, 1982, McNeill & Hamilton, 2003, Donlan & Costerton, 2002). Moreover, several long-term colonizers in humans like *P. aeruginosa*, *S. aureus*, *S. epidermis*, *C. albicans*, *H. influenzae* and *E. coli* grow as extracellular or intracellular biofilms inside the cell, on the tissues, or on medical implantation devices (Blankenship & Mitchell, 2006, Anderson *et al.*, 2004, Davies, 2002, Fey & Olson, Foreman & Wormald, Post, 2001). Furthermore, evidence of direct association between chronic persistence and biofilm formation is found in *S. epidermis* through mutation in a single gene that disrupted both phenotypes (Vuong *et al.*, 2004).

The mechanisms of biofilm formation are primarily investigated in genetically tractable species like *B. subtilis*, *Vibrio spp*. and *Pseudomonas* spp. (Kolter & Losick, 1998, O'Toole *et al.*, 1999, Hall-Stoodley & Stoodley, 2002). Despite the distinction in their specific genetic requirements and structural constituents, biofilms of each species are formed through common developmental mechanisms that involve surface attachment, cell-to-cell communication, and synthesis of extracellular matrix (ECM), which encapsulates the resident cells (Kolter & Losick, 1998, Hall-Stoodley & Stoodley, 2002, Hogan & Kolter, 2002,

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

Fig. 1. Representation of the data published by Jindani et al. (4), showing the pattern of

The hallmark of a successful pathogen is to colonize the host for a long period of time against the challenges of host immune system, and persist against the antibiotics pressure. Although numerous bacterial and fungal pathogens including *M. tuberculosis* easily qualify for this category, the question as to how they establish such infections remains unanswered

However, a large and ever-growing body of evidence provide a compelling argument that the persistence of most, if not all, microbial species in general is achieved through their ability to grow in self-organized, surface associated, sessile communities called biofilms (Costerton et al., 1999, Fux et al., 2005, Hall-Stoodley et al., 2004, Kolter & Greenberg, 2006, Marrie *et al.*, 1982, McNeill & Hamilton, 2003, Donlan & Costerton, 2002). Moreover, several long-term colonizers in humans like *P. aeruginosa*, *S. aureus*, *S. epidermis*, *C. albicans*, *H. influenzae* and *E. coli* grow as extracellular or intracellular biofilms inside the cell, on the tissues, or on medical implantation devices (Blankenship & Mitchell, 2006, Anderson *et al.*, 2004, Davies, 2002, Fey & Olson, Foreman & Wormald, Post, 2001). Furthermore, evidence of direct association between chronic persistence and biofilm formation is found in *S. epidermis* through mutation in a single gene that disrupted both phenotypes (Vuong *et al.*, 2004).

The mechanisms of biofilm formation are primarily investigated in genetically tractable species like *B. subtilis*, *Vibrio spp*. and *Pseudomonas* spp. (Kolter & Losick, 1998, O'Toole *et al.*, 1999, Hall-Stoodley & Stoodley, 2002). Despite the distinction in their specific genetic requirements and structural constituents, biofilms of each species are formed through common developmental mechanisms that involve surface attachment, cell-to-cell communication, and synthesis of extracellular matrix (ECM), which encapsulates the resident cells (Kolter & Losick, 1998, Hall-Stoodley & Stoodley, 2002, Hogan & Kolter, 2002,

*M. tuberculosis* clearance in patients treated with isoniazid and rifampicin.

**4. Chronic infections, bacterial persistence and biofilms** 

for most species.

for a comprehensive understanding of its persistence.

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 antibiotics (Mah & O'Toole, 2001).

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

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 microenvironments that facilitate extensive phenotypic diversity.
