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

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

Biofilms of *Mycobacterium tuberculosis*: New Perspectives of an Old Pathogen 187

Fig. 2. A. Growth of *M. tuberculosis* biofilms on the liquid-air interface in detergent-free Sauton's medium. B. Insertion of a mariner transpososn (Himar) in Rv1013 abolishes the formation of biofilms, although the growth of the mutant in planktonic state remains

Fig. 3. Presence of rifampicin tolerant persisters is significantly higher in biofilm populations of *M. tuberculosis* than in their planktonic counterparts, as reported by Ojha et al. (68).

morphotypes in detail, with equal focus on both the tissue structures as well as bacterial growth (Canetti, 1955). The goal of the undertaking was to bridge the partition between immunopathology and bacteriology of tuberculosis that a student of the disease was always confronted with. This partition ironically continues to be a relevant issue in questions as to how and where bacilli persist in the both latent and active TB. In a recent attempt to locate the bacilli persisting after antibiotic treatment, Orme and colleagues observed the persisters as microcolonies in the acellular rim of granulomas (Lenaerts *et al.*, 2007). Although it was

unaltered (68).

mutation in one of the non-essential chaperone, of *M. smegmatis* specifically retarded the maturation stages of pellicle formation, observed at 4- and 5-day of incubation, without affecting early attachment and growth during first three days of incubation (Ojha *et al.*, 2005). The mutant was also indistinguishable from its wild-type parent in planktonic growth. The maturation defect of the mutant was linked to defective synthesis in mycolic acids as a consequence of the loss of a KasA (enzyme involved in mycolic acid biosynthesis) interaction, which is induced in this phase of wild-type culture (Ojha et al., 2005). The regulated synthesis of mycolic acids was surprising because it is highly abundant in the cell wall, although it is consistent with the subsequent observation of induced synthesis of extracellular free mycolic acids during the maturation stage of the pellicles (Ojha *et al.*, 2010). The free mycolic acids (FM) are released through regulated hydrolysis of mycolyl esters of Trehalose, Trehalose 6',6' dimycolate (TDM), and by cutinase-like serine esterase (Ojha et al., 2010), although other mycolyl esters could also contribute to the FM pool through similar mechanism. One possible candidate could be mycolyl diacyl glycerol (MDAG), a mycolyl ester of glycerol, which is also found in low abundance in impaired biofilms of an *lsr2* mutant of *M. smegmatis* (Chen *et al.*, 2006). The accumulation of FM is likely facilitated through a three-step mechanism: 1) mediated upregulation in *de novo* synthesis of the nascent mycolic acids, 2) processing of nascent mycolic acids into a subset of mycolyl esters through housekeeping mechanisms, and 3) hydrolysis of these mycolyl esters through substrate-specific esterases. The elevated levels of extracellular free mycolic acids during the maturation of *M. smegmatis* pellicles is also consistent with the obvious waxy appearance of the structures, and thus could likely constitute the structure component of the ECM. The significance of *M. smegmatis* growth in pellicles is further highlighted by distinct phenotypes of the bacilli in these structures that are absent in planktonic cell suspension. For example, increased intercellular transfer of genetic materials, and extraordinary tolerance to anti-TB drugs (Ojha et al., 2010, Nguyen *et al.*, 2010). Most importantly, the fundamental properties of mycobacterial pellicles are conserved in *M. tuberculosis* when grown in detergent-free media under defined condition (Ojha et al., 2008). The pathogenic species not only has specific genetic requirement for forming the pellicles (Fig. 2A and B), but also produces large abundance of free mycolic acids in the structure, which expectedly harbor large numbers of drug tolerant bacilli (Ojha et al., 2008) (Fig. 3). Taken together, these *in vitro* studies strongly support the possibility that surface-associated multicellular structures of mycobacteria have all the characteristics of biofilms, developing through distinct growth phases, having specific genetic requirements, and conferring high tolerance to antibiotics. Despite a recent surge in understanding the multicellular structures of mycobacteria, multicellular structures of *M. tuberculosis* in the host have been difficult to define. This in part can be attributed to the conventional image of *M. tuberculosis* as an intracellular pathogen living in phagosome, which is too restrictive for exuberant growth of the pathogen in multicellular structure. However, while *M. tuberculosis* might be restricted to the phagosome in early stages of infection, at later t imes, especially when lesions contain liquefied caseum and when patients are highly infective, it is likely that many of the bacilli experience an extracellular environment. In a comprehensive histopathological study of TB lesions from 1,500 autopsies, Geroges Canetti documented several lesions including open cavities that had numerous extracellular bacilli growing in multicellular structures (Canetti, 1955). Interestingly, in this 7-year study Canetti microscopically analyzed lesions of various

mutation in one of the non-essential chaperone, of *M. smegmatis* specifically retarded the maturation stages of pellicle formation, observed at 4- and 5-day of incubation, without affecting early attachment and growth during first three days of incubation (Ojha *et al.*, 2005). The mutant was also indistinguishable from its wild-type parent in planktonic growth. The maturation defect of the mutant was linked to defective synthesis in mycolic acids as a consequence of the loss of a KasA (enzyme involved in mycolic acid biosynthesis) interaction, which is induced in this phase of wild-type culture (Ojha et al., 2005). The regulated synthesis of mycolic acids was surprising because it is highly abundant in the cell wall, although it is consistent with the subsequent observation of induced synthesis of extracellular free mycolic acids during the maturation stage of the pellicles (Ojha *et al.*, 2010). The free mycolic acids (FM) are released through regulated hydrolysis of mycolyl esters of Trehalose, Trehalose 6',6' dimycolate (TDM), and by cutinase-like serine esterase (Ojha et al., 2010), although other mycolyl esters could also contribute to the FM pool through similar mechanism. One possible candidate could be mycolyl diacyl glycerol (MDAG), a mycolyl ester of glycerol, which is also found in low abundance in impaired biofilms of an *lsr2* mutant of *M. smegmatis* (Chen *et al.*, 2006). The accumulation of FM is likely facilitated through a three-step mechanism: 1) mediated upregulation in *de novo* synthesis of the nascent mycolic acids, 2) processing of nascent mycolic acids into a subset of mycolyl esters through housekeeping mechanisms, and 3) hydrolysis of these mycolyl esters through substrate-specific esterases. The elevated levels of extracellular free mycolic acids during the maturation of *M. smegmatis* pellicles is also consistent with the obvious waxy appearance of the structures, and thus could likely constitute the structure component of the ECM. The significance of *M. smegmatis* growth in pellicles is further highlighted by distinct phenotypes of the bacilli in these structures that are absent in planktonic cell suspension. For example, increased intercellular transfer of genetic materials, and extraordinary tolerance to anti-TB drugs (Ojha et al., 2010, Nguyen *et al.*, 2010). Most importantly, the fundamental properties of mycobacterial pellicles are conserved in *M. tuberculosis* when grown in detergent-free media under defined condition (Ojha et al., 2008). The pathogenic species not only has specific genetic requirement for forming the pellicles (Fig. 2A and B), but also produces large abundance of free mycolic acids in the structure, which expectedly harbor large numbers of drug tolerant bacilli (Ojha et al., 2008) (Fig. 3). Taken together, these *in vitro* studies strongly support the possibility that surface-associated multicellular structures of mycobacteria have all the characteristics of biofilms, developing through distinct growth phases, having specific genetic requirements, and conferring high tolerance to antibiotics. Despite a recent surge in understanding the multicellular structures of mycobacteria, multicellular structures of *M. tuberculosis* in the host have been difficult to define. This in part can be attributed to the conventional image of *M. tuberculosis* as an intracellular pathogen living in phagosome, which is too restrictive for exuberant growth of the pathogen in multicellular structure. However, while *M. tuberculosis* might be restricted to the phagosome in early stages of infection, at later t imes, especially when lesions contain liquefied caseum and when patients are highly infective, it is likely that many of the bacilli experience an extracellular environment. In a comprehensive histopathological study of TB lesions from 1,500 autopsies, Geroges Canetti documented several lesions including open cavities that had numerous extracellular bacilli growing in multicellular structures (Canetti, 1955). Interestingly, in this 7-year study Canetti microscopically analyzed lesions of various

Fig. 2. A. Growth of *M. tuberculosis* biofilms on the liquid-air interface in detergent-free Sauton's medium. B. Insertion of a mariner transpososn (Himar) in Rv1013 abolishes the formation of biofilms, although the growth of the mutant in planktonic state remains unaltered (68).

Fig. 3. Presence of rifampicin tolerant persisters is significantly higher in biofilm populations of *M. tuberculosis* than in their planktonic counterparts, as reported by Ojha et al. (68).

morphotypes in detail, with equal focus on both the tissue structures as well as bacterial growth (Canetti, 1955). The goal of the undertaking was to bridge the partition between immunopathology and bacteriology of tuberculosis that a student of the disease was always confronted with. This partition ironically continues to be a relevant issue in questions as to how and where bacilli persist in the both latent and active TB. In a recent attempt to locate the bacilli persisting after antibiotic treatment, Orme and colleagues observed the persisters as microcolonies in the acellular rim of granulomas (Lenaerts *et al.*, 2007). Although it was

Biofilms of *Mycobacterium tuberculosis*: New Perspectives of an Old Pathogen 189

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