**3.3 Growth characteristics and colonial morphology**

In contrast to classical tubercle bacilli, we found that L-form variants, obtained after nutrient starvation stress of *M. tuberculosis in vitro,* grew and developed colonies phenomenally faster, mimicking rapidly replicating bacteria.

The morphology of growths underwent progressive changes, which resulted in formation of typical L-form colonies with "fried egg" appearance (Fig.7 b, c).

As pointed out by other authors, dark centers of "fried egg" colonies usually consist of dense granular elements, which are deeply embedded in the medium but at the periphery of the colony large pleomorphic bodies are frequently found (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981). The shape of L-form colonies resulted from the variety of individual structural units and the way that they divided (Mattman, 2001). It should be pointed out that fully developed L-type colonies appeared between 36 and 48 hours after plating on Middlebrook semisolid agar, in contrast to control *M. tuberculosis* microcolonies.

Fig. 6. Morphological phases during L-form conversion and reversion of mycobacteria.

Spacer sequences and IS *6110* PCR, verified them as *M. tuberculosis* (n. d.). DNA sequencing analysis is currently in progress (n. d.).We consider that the invisible Lconversion phase is followed by a state of active reproduction of non- acid fast and nonrecognizable as mycobacteria L-forms usually with coccoid morphology. Taken together, these data may argue that the curious morphology and growth characteristics of mycobacterial L-forms, their extremely different habit of existence define them as specific type of unrecognizable and hidden persisters. As seen in Fig.6, L-form conversion cycle of mycobacteria is schematically outlined with emphasis on ability of different L-structures to form colonies. In this sense, L-form persistence phenomenon substantially differs from the current understanding for latency as persistence of few ''non-replicating''or

In contrast to classical tubercle bacilli, we found that L-form variants, obtained after nutrient starvation stress of *M. tuberculosis in vitro,* grew and developed colonies phenomenally

The morphology of growths underwent progressive changes, which resulted in formation of

As pointed out by other authors, dark centers of "fried egg" colonies usually consist of dense granular elements, which are deeply embedded in the medium but at the periphery of the colony large pleomorphic bodies are frequently found (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981). The shape of L-form colonies resulted from the variety of individual structural units and the way that they divided (Mattman, 2001). It should be pointed out that fully developed L-type colonies appeared between 36 and 48 hours after plating on Middlebrook semisolid agar, in contrast to control *M. tuberculosis*

**Stress induction of L-forms Reversion** 

**Transitional incomplete reversion**  non - acid fast rod-shaped forms that form smooth

colonies

**Complete reversion**  to acid-fast, slow growing mycobacteria formation of typical rough colonies

Fig. 6. Morphological phases during L-form conversion and reversion of mycobacteria.

**L-phase variants of mycobacteria** 

''dormant'' bacteria.

microcolonies.

 **"Invisible"** cryptic state of Lforms that do not form colonies: giant L-structures (filaments or large "mother" L-bodies) and filterable Lgranules

**3.3 Growth characteristics and colonial morphology** 

typical L-form colonies with "fried egg" appearance (Fig.7 b, c).

**"Visible"**  L-phase growth of non- acid fast polymorphic or coccoid cells resulting in formation of "fried eggs" or smooth colonies

faster, mimicking rapidly replicating bacteria.

We suggest that the lack of cell walls and easier permeation of nutrients is the reason for the unique ability of mycobacterial L- forms to grow faster in comparison to classical tubercle bacilli. Pla Y Armengol (1931) found that a large inoculum of tubercle bacilli grows rapidly on all routine media, appearing as large L-body spheres and also vegetated mycelia. In our study, L-form variants were adapted without difficulties to grow on conventional nutrient agar. Light and electron microscopy also provided interesting results about the appearance of non-acid fast coccoid cell morphology of stressed *M. tuberculosis*, that support observations of other authors. The appearance of non-acid fast coccoids in cultures of mycobacteria has been reported by others in the beginning of the last century but the phenomenon was not clearly explained and proven at that time (Csillag, 1964; Juhasz, 1962; Xalabander, 1958;). More surprising was the fact that mycobacterial coccoid L-forms not only mimicked the morphology of staphylococci or other coccus- shaped bacteria, but also exhibited extremely rapid growth and colonial development in contrast to classical TB bacilli (n. d.). Coccoid cells were initially mistaken by us as contaminants, but the specific DNA testing (amplification of 16SrRNA gene fragment, 16S-23S rRNA gene Internal Transcribed Spacer sequences, IS*6110* PCR and DNA sequencing analysis) identified them as *M. tuberculosis* (n. d.). We suppose that non-acid fast coccoid L-form variants of mycobacteria resulted probably from the more regular mode of multiplication, synchronization and stabilization of L-form cells under specific condition of cultivation. Thus, it can be presumed why such coccoid forms of *M. tuberculosis* remain often unrecognized or are mistaken for contaminants.

Fig. 7. Light microscopy of (a) control *M. tuberculosis* rough microcolony and (b, c) typical "fried eggs" shaped colonies of *M.tuberculosis* L-forms obtained after nutrient starvation stress (n. d.).

a. b. c.

Standard plating techniques are often inadequate for accurate enumeration of microbial dormant forms, because some of them may be in a "nonculturable" state (Shleeva et al., 2010). When it comes to L-forms**,** they are considered to be both "difficult-to-cultivate" and "difficultto-identify". Because of their altered morphology and fully changed bacterial life cycle, Lforms are difficult to be identified in clinical materials. The isolation of arising *in vivo* L-forms is generally possible only with special procedures ensuring their enrichment and resuscitation to actively growing state i.e. having an ability to form colonies (Michailova et al., 2000a; Zhang et al., 2001; Zhang, 2004). The use of specially supplemented liquid and semisolid media, as well special techniques, like so called "blind" passages, are absolutely necessary for isolation of L-forms from specimens (Michailova et al., 2005; Markova et al., 2008a).

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 203

L-forms within natural populations of other bacteria as well as about relations of concurrence and interference between them under different conditions (Boris *et al.*, 1969; Fodor and Roger, 1966). Extreme morphological plasticity of bacteria has been found to provide survival advantages (Justice *et al*., 2008; Young, 2007). It is assumed that L-forms occur along with resistance to factors that trigger their appearance (Prozorovski, 1981).

We found that under stress *in vitro* and *in vivo* (Markova et al, 2008a) the balance within mycobacteria was shifted in favor of cell wall deficient forms and the population continued to exist, replicating predominantly as L-forms. Our *in vitro* experiments aimed to induce Lconversion of *M. tuberculosis,* by means of nutrient starvation stress (n. d.). Once the process in favor of L-form development was induced and shifted, further selective separation of Lform variants was made, based on the unique ability of mycobacterial L- forms to grow faster in comparison to classical tubercle bacilli due to the lack of cell walls and easier permeation of nutrients. Selection of mycobacterial L-forms was achieved technically through transfers of stressed mycobacterial cultures at weekly intervals on semisolid Middlebrook agar. Due to their growth advantage, mycobacterial L-form variants became the prevailing subpopulation, overgrowing classical TB bacilli during the performed five passages, which resulted in isolation of L-form cultures. As has been demonstrated in our previous study, similar L-form transformation of *E. coli* was found to appear under conditions of starvation and, more surprisingly, after lethal heat stress (Markova et al., 2010). However, it has been found that cell wall deficient forms of *E. coli* developed slower than classical walled forms. In contrast with interactions between both subpopulations during L-form transformation of *E. coli*, we recognized the opposite relations between classical and L-forms in mycobacteria, which were strongly influenced by the special

biochemical structure and physiology of TB bacilli.

**4. Formation and persistence of mycobacterial L-forms in vivo** 

Animal models for the study of tuberculosis include guinea pigs, mice, rabbits and nonhuman primates. Despite the difficulty in modeling human latency in experimental animals, the understanding of both host and microbial factors that contribute to the establishment and maintenance of a persistent *M. tuberculosis* infection has progressed and the information gathered is pertinent to human latent tuberculosis (Flynn & Chan, 2001). Formation of *M. tuberculosis* L-forms *in vivo* were demonstrated by means of biological experiments on guinea pigs (Li, 1990; Markova et al., 2008b ; Ratnam &Chandrasekhar, 1976;

In our study, we established a rat model of experimental tuberculosis that produces mycobacterial cell-wall deficient forms *in vivo* (Markova et al, 2008a). Although rats are not a common animal model for TB research, we attempted, on basis of our previous experience with other bacterial L- form experimental infections (Markova et al, 1997; Michailova et al., 2000), to use the capability of these animals to exhibit high innate resistance to infections, thus ensuring inhibition of classical bacterial forms and inducing the occurrence of cell-wall deficient forms. After intraperitoneal and intranasal infection with *M. tuberculosis*, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes and bronchoalveolar and peritoneal lavage liquid were taken and plated simultaneously on Löwenstein-Jensen medium or inoculated into specially supplemented for L-forms Dubos broth at weekly intervals over five weeks. Mycobacterial L-form cultures were isolated throughout

Snitinskaia et al., 1990;), mice (Belianin et al., 1997) and rats (Markova et al., 2008a).

#### **3.4 Yin-Yang hypothesis for co-existence of classical and L- forms within natural mycobacterial population**

The Yin-Yang hypothesis is based on the idea that classical and cell wall deficient forms coexist within natural mycobacterial populations. The Chinese concept of the complementary alternating forces of Yin and Yang provides opportunities to better understand the natural phenomenon of heterogeneity and correspondence between both subpopulations in mycobacteria. The Yin-Yang point of view, suggesting the hypothesis for coexistence of classical and cell wall deficient forms, is illustrated in Fig.6.

Fig. 8. Alternating "Yin-Yang"–life phases of classical walled bacteria and cell wall deficient L-forms: "Yang" - monomorphic population of classical rod shaped bacteria under optimal conditions ensuring yield and nutrients for growth and cell division; "Yin"- polymorphic population of L-forms. Polymorphism ensures survival advantages and arsenal of various notoriously resistant to environmental assaults L-form structures under unfavorable conditions.

The morphological diversity within bacterial populations is often related to heterogeneous environments observed under natural conditions. Population-based morphological variability and cell wall deficiency of *M. tuberculosis* might be considered as a natural phenomenon ensuring the adaptive strategy of this pathogen for environmental change (Markova, 2009; Mattman, 2001). By making use of our Yin-Yang concept, by utilizing available scientific data on this subject and our own findings, we try to figure out how classical walled and cell wall deficient subpopulations interact under different conditions. We found that both classical walled and cell wall deficient L-forms coexist within clinical strains of *M. tuberculosis* freshly isolated from sputum of patients has been demonstrated in our study (Michailova et al., 2005). This finding supports the concept that natural mycobacterial population usually consists of prevalent classical forms and small numbers of L- forms. There is data reporting about coexistence of classical walled and cell wall deficient

The Yin-Yang hypothesis is based on the idea that classical and cell wall deficient forms coexist within natural mycobacterial populations. The Chinese concept of the complementary alternating forces of Yin and Yang provides opportunities to better understand the natural phenomenon of heterogeneity and correspondence between both subpopulations in mycobacteria. The Yin-Yang point of view, suggesting the hypothesis for coexistence of

Fig. 8. Alternating "Yin-Yang"–life phases of classical walled bacteria and cell wall deficient L-forms: "Yang" - monomorphic population of classical rod shaped bacteria under optimal conditions ensuring yield and nutrients for growth and cell division; "Yin"- polymorphic population of L-forms. Polymorphism ensures survival advantages and arsenal of various notoriously resistant to environmental assaults L-form structures under unfavorable

The morphological diversity within bacterial populations is often related to heterogeneous environments observed under natural conditions. Population-based morphological variability and cell wall deficiency of *M. tuberculosis* might be considered as a natural phenomenon ensuring the adaptive strategy of this pathogen for environmental change (Markova, 2009; Mattman, 2001). By making use of our Yin-Yang concept, by utilizing available scientific data on this subject and our own findings, we try to figure out how classical walled and cell wall deficient subpopulations interact under different conditions. We found that both classical walled and cell wall deficient L-forms coexist within clinical strains of *M. tuberculosis* freshly isolated from sputum of patients has been demonstrated in our study (Michailova et al., 2005). This finding supports the concept that natural mycobacterial population usually consists of prevalent classical forms and small numbers of L- forms. There is data reporting about coexistence of classical walled and cell wall deficient

**3.4 Yin-Yang hypothesis for co-existence of classical and L- forms within natural** 

classical and cell wall deficient forms, is illustrated in Fig.6.

**mycobacterial population** 

conditions.

L-forms within natural populations of other bacteria as well as about relations of concurrence and interference between them under different conditions (Boris *et al.*, 1969; Fodor and Roger, 1966). Extreme morphological plasticity of bacteria has been found to provide survival advantages (Justice *et al*., 2008; Young, 2007). It is assumed that L-forms occur along with resistance to factors that trigger their appearance (Prozorovski, 1981).

We found that under stress *in vitro* and *in vivo* (Markova et al, 2008a) the balance within mycobacteria was shifted in favor of cell wall deficient forms and the population continued to exist, replicating predominantly as L-forms. Our *in vitro* experiments aimed to induce Lconversion of *M. tuberculosis,* by means of nutrient starvation stress (n. d.). Once the process in favor of L-form development was induced and shifted, further selective separation of Lform variants was made, based on the unique ability of mycobacterial L- forms to grow faster in comparison to classical tubercle bacilli due to the lack of cell walls and easier permeation of nutrients. Selection of mycobacterial L-forms was achieved technically through transfers of stressed mycobacterial cultures at weekly intervals on semisolid Middlebrook agar. Due to their growth advantage, mycobacterial L-form variants became the prevailing subpopulation, overgrowing classical TB bacilli during the performed five passages, which resulted in isolation of L-form cultures. As has been demonstrated in our previous study, similar L-form transformation of *E. coli* was found to appear under conditions of starvation and, more surprisingly, after lethal heat stress (Markova et al., 2010). However, it has been found that cell wall deficient forms of *E. coli* developed slower than classical walled forms. In contrast with interactions between both subpopulations during L-form transformation of *E. coli*, we recognized the opposite relations between classical and L-forms in mycobacteria, which were strongly influenced by the special biochemical structure and physiology of TB bacilli.
