**9. Cell shapes during dormancy or under limited conditions**

The morphological variations in tubercle bacilli become evident when the culture medium was poor. These changes were first reported by Koch himself. In his paper on the "discovery

developed dense bodies in polar positions and in some organisms a number of smaller are also seen scattered among the cellular units and apparently associated with them. The final stage in the reproductive cycle led to a massive production of small rods. At this phase the filaments suddenly break down into masses of short rods which elongate to form the new generation and the cycle is complete. Under electron microscope, it was seen that the filaments were quite separate, and there was no true branching and that the mycelia appearance was produced because the filaments often remained stuck together. In another study, Mizuguchi Y *et al* (1985) showed β-Lactam antibiotics at low concentration induced filamentous cells in the *M. avium-intracellular* complex. Although, the mechanisms of induction of filamentous cells appeared to be different according to the drugs used. Ampicillin induces filaments by inhibiting the septation in a manner similar to its effect on *E. coli,* whereas cephazolin induces filaments but does not inhibit septation. In *M. tuberculosis,* branches were first seen as a small bud that does not grow to any appreciable size before breaking off as a separate cell. Few studies suggested that *M. tuberculosis* grows from the ends of bacilli and not along the length of the cylinder as seen in other well-characterized rod shape bacteria (Thanky *et al*., 2007). This might be true for susceptible isolates, but recently Farnia *et al* (2010) showed that in highly drug resistance strains i.e., XDR-TB and Totally or Extremely drug resistant isolates (TDR or XXDR-TB), branches produce along the cylinder. In fact, about 20 -24% of cells in XDR and XXDR-TB

Fig. 3. Transmission Electron Microscopy shows Y-Shape *M. tuberculosis* at exponential

The morphological variations in tubercle bacilli become evident when the culture medium was poor. These changes were first reported by Koch himself. In his paper on the "discovery

**9. Cell shapes during dormancy or under limited conditions** 

bacilli were dividing by branching, respectively.

phase of growth

of the cause of tuberculosis", he described that "under certain conditions, some bacilli contain several spores, in most cases there are two to four of them; oval in form, they are distributed, in uniform intervals, along the axis of the bacilli(1882). Following Koch discovery, Malassez and Vignal (1883) had described, the small "coccoid bodies " which cause tuberculosis infection and named them as cell wall deficient forms (CWD-forms) of tuberculosis. Later on, Spengler (1903, 1905), were among the first scientist who could demonstrated that in older cultures and frequently in sputa, apparently in response to adverse environmental conditions, the smooth cell takes on a fragmented appearance. Much (1907) was able to reproduce granules in the inside of the bacilli as well as scattered around them. These granules, according to Much, cannot be stained by the Ziehl- Neelsen technique but may generate new tubercle bacilli. Later on 1909, Fontes revealed how he had applied double staining to the bacilli, namely Ziehl- Neelson's carbolfuchsin staining and the Gram treatment. In this way he tried to differentiate the pathogenic tubercle bacilli, containing Much granules, from the apathogenic ones without these granules. In 1910, Fontes described the multiplication through division of these granules in the inside of a cell and on its outside and applied the term "virus" to this formation. Fontes described the application to the tubercule bacillus of the well –known method of separating the virus from the substrate by filtering the material through a bacterial filter. He inoculated a guinea pig with the filtered caseous material and transplanted the organs of this animal into a fresh one. When after five months of observation the animal was killed, the autopsy revealed the infiltration of round cells, granules, and occasional acid-fast bacilli in the lymph nodes and the lungs. After years of oblivion, the early works of Fontes were rediscovered by Vandremer (1923). He repeated the Fontes filtration experiments and confirmed the development of acid –fast bacilli on media and in animals inoculated with these filtrates. Calmette (1926) advanced the theory on the role of the the tuberculosis " ultra-virus" in the development of certain forms of the diseases. However, Negre *et al* (1933) denied the existence of filterable forms of the mycobacteria. Few years later, Vera and Rettger (1939) studied four strains of *M. tuberculosis*(hominis), "Koch", 607, 75 and H37 in micro-culture by Hill hanging block technique. This method was employed to permit observation of individual cells and their progeny over long periods of time using lucida drawings camera. They could demonstrate various forms which have been described in the literature at one time or another. When they cut off air supply, different variants developed very soon. The bacilli swelled slightly, the cytoplasm become less clear and smooth. The swelling commonly occurred at the ends of cells, so the clubs and dumbbell shapes were formed; cells often became spoon shaped. These swollen structures became increasingly refractive and more sharply delimited, until finally there was a definite superficial resemblance to spores. At the similar time, the ability of the tubercle bacillus to survive environmental hardship in culture was documented by Corper and Cohn in a study published in 1933. In another study, McCune and other colleagues (1965, 1966), showed the capacity of tubercle bacilli to survive in mouse tissue after sterilization. In this model, out bred mice were infected intravenously with 105 colonyforming units of the H37Rv strain of *M. tuberculosis*. They were immediately treated for a period of 12 weeks with the antimycobacterial drugs isonizid (INH) and pyrazinamide (PZA). For 4-6 week period after withdrawal of therapy, the mice showed no evidence of cultivable tubercle bacilli (sterile state). But, 12 weeks after INH and PZA treatment was withdrawn, one-third of the mice developed full-blown active TB, with nearly two-thirds

Morphological Characterization of *Mycobacterium tuberculosis* 159

obtained by Wayne and Sramek (1994) as dormant because they maintained a high viability and developed sensitivity to metronidazole when anaerobic, thus indicating active metabolism. Therefore, from large accumulated data that found in literature, it become clear that *M. tuberculosis* can adapt rapidly to changing environment inside and outside the host (Parrish *et al*., 1998; Cardona, 2009; Rustad *et al*., 2009). These capacities will allow the tubercle bacilli to survive for long time in a dormant state in the lung tissue. Recently, Peyron *et al* (2008) developed an *in vitro* model of human tuberculosis granulomas. In this model granuloma-specific cell types and their modulation by tubercle bacilli were characterized. More recently, the complete morphological changes that occurs in tubercle bacilli under hypoxic conditions viewed under AFM (every 90 days for 48 months) (Velayati *et al*., 2011). The morphological adaptation classified into two categories; First was temporary adaptation (from 1 to 18 months of latency) in which cells undergoing thickening of cell wall (20.5±1.8 nm versus 15.2±1.8 nm, P<0.05), formation of ovoid cells by "folding phenomena"(65-70%), size reduction (0.8± 0.1 µm versus 2.5±0.5

Fig. 4. Atomic force Microscopy shows *M. tuberculosis* under 8 months hypoxic condition. The bacilli becomes round and developed a thickened cell-walls (shows by arrows)

A second feature include changes that accompany development of specialized cells (from 18 to 48 months of latency) i.e., production of spore like cells (0.5 ± 0.2 µm) and their progeny (filterable non -acid fast forms; 150 to 300 µm in size figure 5). Using AFM, they could demonstrate that the filterable non-acid fast forms of bacilli are produced from spore –like cells. These cells were metabolically active and increased their number by symmetrical typing of division and could be stain by gram staining. Inoculation of these cells could induce active tuberculosis in mice. Although, it is important to determine how closely the

µm), and budding type of cell division (20-25%) (figure 4).

displaying disease after 24 weeks. Csillag (1962, 1963, and 1964) considered Mycobacteria as dimorphic organisms in the same sense as are some pathogenic fungi, for instance, *Histoplasma capsulatum*. The usual acid fast form of the mycobacteria was termed 'form I' and the form which was not acid fast was termed 'form 2'. When form 2 grown in digest broth, form 2 strains produced cocci which continued to multiply by binary fission and bud formation (Csillag, 1964).

These forms were not produced by mycobacteria grown in rich media such as nutrient broth; Martin's digest broth, yeast extract and Lab-Lemo beef extract. One year later, Stewart-Tull (1965) isolated two forms of mycobacteria and mycococci from *M. phlei* .Nyka W in 1963, described them as "chromophobic tubercle bacilli" in the lungs of patients treated by drugs in association with surgery. This organism morphologically were similar to the acid- fast bacilli, but do not stain with either carbolfuchsin or the counter stains when applied by the classic Ziehl-Neelsen technique or with any other aniline dye. In continuation of his work, he submitted the culture of *M. tuberculosis*, *M. kansasii*, and *M. phlei* to starvation. As a result they lost first their acid fastness, but in this chromophobic state, they survived for at least 2 years, and after that time, produced cultures of acid fast bacilli when transferred onto nutrient media. Since these *in-vitro* bacilli could recover their original biological properties, it was concluded that those bacilli in the lung could also become reactivated and cause a relapse of the disease. Some scientists regard the filterable forms of mycobacteria as being analogous to the so –called L-forms of the other bacterial genera as they also pass through filters (Thacore and Willett, 1963). Some other scientists believe that development of the L-form is a mutation process, while development of the filterable forms is an adaptation of the microorganisms to enable them to multiply in unfavorable (Imaeda, 1974; Mattman, 1970: Ratnam and Chandrasekhar, 1976). In this regards, Takahashi (1979), reported that tubercle bacilli in caseous lesions seems to be non acid fast, gram negative granules which may revert into acid fast rods, when the caseous lesion begins to liquefy and form tuberculous cavity. Similarly, khomenko and colleagues (1987) showed ultra-fine forms of *M. tuberculosis* in the walls of open cavities in the lungs of experimental animals by electron microscopy. These invisible forms of *M. tuberculosis* are able to revert to the typical bacterial forms. The initial stage of this process is accompanied by the formation of coccoid forms of mycobacteria that can be detected when material is inoculated on to semi-synthetic medium with 10% plasma and by microscopy of the sediment. Lawrence Wayne (1994) postulated that bacilli recovered from granulomatous lesions had adapted to a relatively oxygen starved environment so that they would be unable to grow in an aerated culture and therefore, may be non-cultivable by traditional culture methods (Wayne and Hayes, 1996). In the Wayne model, cultures of the bacterium are subjected to gradual self-generated oxygen depletion by incubation in sealed stirred tubes. Upon the slow shift of aerobic growing *M. tuberculosis* to anaerobic conditions, the culture is able to adapt and survive anaerobiosis by shifting down to a state of nonreplicating persistence. Wayne L showed two phase of growth in mycobacterium under limited oxygen; initially when the level of drops and the turbidity increased in culture tubes (NRP-1) and in anaerobic phase when there is no oxygen and no division (NRP-2). Wayne model was a break through in understanding what may happen to tubercule bacilli in necrotic material (Wayne and Lin, 1982). Although, Kaprelyants *et al* (1993) did not consider the bacilli

displaying disease after 24 weeks. Csillag (1962, 1963, and 1964) considered Mycobacteria as dimorphic organisms in the same sense as are some pathogenic fungi, for instance, *Histoplasma capsulatum*. The usual acid fast form of the mycobacteria was termed 'form I' and the form which was not acid fast was termed 'form 2'. When form 2 grown in digest broth, form 2 strains produced cocci which continued to multiply by binary fission and bud

These forms were not produced by mycobacteria grown in rich media such as nutrient broth; Martin's digest broth, yeast extract and Lab-Lemo beef extract. One year later, Stewart-Tull (1965) isolated two forms of mycobacteria and mycococci from *M. phlei* .Nyka W in 1963, described them as "chromophobic tubercle bacilli" in the lungs of patients treated by drugs in association with surgery. This organism morphologically were similar to the acid- fast bacilli, but do not stain with either carbolfuchsin or the counter stains when applied by the classic Ziehl-Neelsen technique or with any other aniline dye. In continuation of his work, he submitted the culture of *M. tuberculosis*, *M. kansasii*, and *M. phlei* to starvation. As a result they lost first their acid fastness, but in this chromophobic state, they survived for at least 2 years, and after that time, produced cultures of acid fast bacilli when transferred onto nutrient media. Since these *in-vitro* bacilli could recover their original biological properties, it was concluded that those bacilli in the lung could also become reactivated and cause a relapse of the disease. Some scientists regard the filterable forms of mycobacteria as being analogous to the so –called L-forms of the other bacterial genera as they also pass through filters (Thacore and Willett, 1963). Some other scientists believe that development of the L-form is a mutation process, while development of the filterable forms is an adaptation of the microorganisms to enable them to multiply in unfavorable (Imaeda, 1974; Mattman, 1970: Ratnam and Chandrasekhar, 1976). In this regards, Takahashi (1979), reported that tubercle bacilli in caseous lesions seems to be non acid fast, gram negative granules which may revert into acid fast rods, when the caseous lesion begins to liquefy and form tuberculous cavity. Similarly, khomenko and colleagues (1987) showed ultra-fine forms of *M. tuberculosis* in the walls of open cavities in the lungs of experimental animals by electron microscopy. These invisible forms of *M. tuberculosis* are able to revert to the typical bacterial forms. The initial stage of this process is accompanied by the formation of coccoid forms of mycobacteria that can be detected when material is inoculated on to semi-synthetic medium with 10% plasma and by microscopy of the sediment. Lawrence Wayne (1994) postulated that bacilli recovered from granulomatous lesions had adapted to a relatively oxygen starved environment so that they would be unable to grow in an aerated culture and therefore, may be non-cultivable by traditional culture methods (Wayne and Hayes, 1996). In the Wayne model, cultures of the bacterium are subjected to gradual self-generated oxygen depletion by incubation in sealed stirred tubes. Upon the slow shift of aerobic growing *M. tuberculosis* to anaerobic conditions, the culture is able to adapt and survive anaerobiosis by shifting down to a state of nonreplicating persistence. Wayne L showed two phase of growth in mycobacterium under limited oxygen; initially when the level of drops and the turbidity increased in culture tubes (NRP-1) and in anaerobic phase when there is no oxygen and no division (NRP-2). Wayne model was a break through in understanding what may happen to tubercule bacilli in necrotic material (Wayne and Lin, 1982). Although, Kaprelyants *et al* (1993) did not consider the bacilli

formation (Csillag, 1964).

obtained by Wayne and Sramek (1994) as dormant because they maintained a high viability and developed sensitivity to metronidazole when anaerobic, thus indicating active metabolism. Therefore, from large accumulated data that found in literature, it become clear that *M. tuberculosis* can adapt rapidly to changing environment inside and outside the host (Parrish *et al*., 1998; Cardona, 2009; Rustad *et al*., 2009). These capacities will allow the tubercle bacilli to survive for long time in a dormant state in the lung tissue. Recently, Peyron *et al* (2008) developed an *in vitro* model of human tuberculosis granulomas. In this model granuloma-specific cell types and their modulation by tubercle bacilli were characterized. More recently, the complete morphological changes that occurs in tubercle bacilli under hypoxic conditions viewed under AFM (every 90 days for 48 months) (Velayati *et al*., 2011). The morphological adaptation classified into two categories; First was temporary adaptation (from 1 to 18 months of latency) in which cells undergoing thickening of cell wall (20.5±1.8 nm versus 15.2±1.8 nm, P<0.05), formation of ovoid cells by "folding phenomena"(65-70%), size reduction (0.8± 0.1 µm versus 2.5±0.5 µm), and budding type of cell division (20-25%) (figure 4).

Fig. 4. Atomic force Microscopy shows *M. tuberculosis* under 8 months hypoxic condition. The bacilli becomes round and developed a thickened cell-walls (shows by arrows)

A second feature include changes that accompany development of specialized cells (from 18 to 48 months of latency) i.e., production of spore like cells (0.5 ± 0.2 µm) and their progeny (filterable non -acid fast forms; 150 to 300 µm in size figure 5). Using AFM, they could demonstrate that the filterable non-acid fast forms of bacilli are produced from spore –like cells. These cells were metabolically active and increased their number by symmetrical typing of division and could be stain by gram staining. Inoculation of these cells could induce active tuberculosis in mice. Although, it is important to determine how closely the

Morphological Characterization of *Mycobacterium tuberculosis* 161

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*in vitro* models correlate to the state of *M. tuberculosis* during latent infection. But, if these models are predictive of human disease, the information they provide in combination with advances in animal models, imaging and analysis will substantially aid in the development of drugs capable of killing tubercle bacilli in altered metabolically states, and possibly shortening the course of TB therapy.

Fig. 5. Atomic force microscopy shows the Latent TB bacilli, after 48 months of latency (Velayati *et al*., 2011).
