**6. Exploring** *M. tuberculosis* **biofilms** *in vivo*

The *in vitro* studies on mycobacterial persistence in biofilms provide a compelling argument that the extracellular multicellular structures of *M. tuberculosis* in liquefied lesions could be primary foci of persister cells. A basic approach in such a study will involve three critical components: a) imaging *M. tuberculosis* bacilli in intact lesions, b) identifying molecular signatures like free mycolic acids of biofilms in multicellular structures of bacilli, and c) genetically correlating persistence with multicellular structures. Imaging bacilli in intact lesions could be a potentially challenging and expensive approach but is important because conventional processing of tissues with harsh organic chemicals used in typical histopathological protocols likely distort the bacillary architecture. Moreover, the modified cell wall of mycobacteria in biofilms could also render them undetectable by acid-fast staining. Confocal Laser Scanning Microscopy (CLSM) of fluorescently marked *M. tubercul*osis in lesions resected from an animal model that closely mimic human infections, like non-human primates, represents an attractive approach to imaging *M. tuberculosis in vivo* biofilms. The imaging studies could then be followed with detection and analysis of extracellular molecules including lipids and proteins that are associated with multicellular structures. Although the abundance of free mycolic acids in biofilms *in vitro* makes this an excellent candidate, the search should remain open, in case different surface molecules are used for cohesion of the structures *in vivo*. Finally, a systematic *in vivo* analysis of genetically defined mutants that fail to form *in vitro* biofilms could be a powerful strategy for gaining mechanistic insights and identifying drug targets that can dismantle the biofilm structure. It is noteworthy that transposon insertion in *pks16* (Rv1013) and *helY* can impair the development of *M. tuberculosis*, although it is unclear whether the effects of genes products are directly on structural formation or indirectly on adaption of resident bacteria within the structure (Ojha et al., 2008). Mutants in the former category could be especially useful for *in vivo* studies avoiding indirect effects of gene products resulting from changes in the morphologies of the structures.

## **7. Conclusions**

Although a short and effective treatment of *M. tuberculosis* infection remains a big challenge to mankind, a solution is unlikely to appear without mechanistic insights into the persistent nature of the pathogen. At the origin of such studies lies a growth model that would reflect the spontaneous behavior of the pathogen. Use of detergents in the process of growing dispersed *in vitro* cultures has arguably misrepresented the physical existence of *M. tuberculosis* in its natural context. In the absence of detergent, the pathogen forms drug tolerant multicellular biofilms, and the complex structures of biofilms undoubtedly hold a treasure of information about the mechanisms that shape their behavior. It is time we focused on these observations to develop new strategies to combat Man's deadliest microbial enemy – *M. tuberculosis*.

#### **8. References**

188 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

not clear in their study whether these bacilli were alive or dead, these are reminiscent of the

The *in vitro* studies on mycobacterial persistence in biofilms provide a compelling argument that the extracellular multicellular structures of *M. tuberculosis* in liquefied lesions could be primary foci of persister cells. A basic approach in such a study will involve three critical components: a) imaging *M. tuberculosis* bacilli in intact lesions, b) identifying molecular signatures like free mycolic acids of biofilms in multicellular structures of bacilli, and c) genetically correlating persistence with multicellular structures. Imaging bacilli in intact lesions could be a potentially challenging and expensive approach but is important because conventional processing of tissues with harsh organic chemicals used in typical histopathological protocols likely distort the bacillary architecture. Moreover, the modified cell wall of mycobacteria in biofilms could also render them undetectable by acid-fast staining. Confocal Laser Scanning Microscopy (CLSM) of fluorescently marked *M. tubercul*osis in lesions resected from an animal model that closely mimic human infections, like non-human primates, represents an attractive approach to imaging *M. tuberculosis in vivo* biofilms. The imaging studies could then be followed with detection and analysis of extracellular molecules including lipids and proteins that are associated with multicellular structures. Although the abundance of free mycolic acids in biofilms *in vitro* makes this an excellent candidate, the search should remain open, in case different surface molecules are used for cohesion of the structures *in vivo*. Finally, a systematic *in vivo* analysis of genetically defined mutants that fail to form *in vitro* biofilms could be a powerful strategy for gaining mechanistic insights and identifying drug targets that can dismantle the biofilm structure. It is noteworthy that transposon insertion in *pks16* (Rv1013) and *helY* can impair the development of *M. tuberculosis*, although it is unclear whether the effects of genes products are directly on structural formation or indirectly on adaption of resident bacteria within the structure (Ojha et al., 2008). Mutants in the former category could be especially useful for *in vivo* studies avoiding indirect effects of gene products resulting from changes in the

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extracellular multicellular structures of bacilli reported in Canetti's study.

**6. Exploring** *M. tuberculosis* **biofilms** *in vivo*

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microbial enemy – *M. tuberculosis*.

**7. Conclusions** 


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**11** 

Nadya Markova

*Bulgaria* 

**Cell Wall Deficiency in Mycobacteria:** 

It is believed that persistence of small populations of *Mycobacterium tuberculosis* in hosts underlies latent tuberculosis. Very little is known about the morphological and physiological nature of tubercle bacilli in latent TB. It is under discussion whether and how tubercle bacilli adapt to latent state and remain alive in face of damaging stressful conditions such as antibiotics and host immune factors. In this respect, cell wall deficiency (existence without rigid walls) in mycobacteria and its occurrence *in vivo* suggests one of the possible pathways by which tubercle bacilli can survive, replicate and persist within the body for a long period, harboring latent tuberculosis with a risk for disease reactivation, in case of reversion to classical TB bacilli upon changes in host immune status. Essentially, cell wall deficiency, or the ability of bacteria to exist as populations of self-replicating forms with defective or entirely missing cell walls (L-forms), is considered an adaptive strategy of

This chapter elaborates on some special aspects of the L-form phenomenon and its importance for discovering new fundamental aspects of TB bacillary morphology and

L-forms were first observed by Emmy Klieneberger-Nobel, in 1945, whose typical "fried eggs"-shaped colonies, duplicating Mycoplasma, were isolated from cultures of *Streptobacillus moniliformis.* The wall-less variants of L-forms she named after the institution

bacteria to survive and reproduce under unfavorable circumstances.

Fig. 1. Emmy Klieneberger-Nobel – the founder of bacterial L-forms.

physiology, as well as understanding the mechanisms of latent tuberculosis.

**1. Introduction** 

**2. History** 

she worked in – England's Lister Institute.

**Latency and Persistence** 

*Institute of Microbiology, Bulgarian Academy of Sciences* 

