**2.2 An effect of lipids on growth and viability of** *Mycobacterium tuberculosis* **H37Rv**

Then we have studied a growth and viability of pathogenic *Mycobacterium tuberculosis*  H37Rv *in vitro* during cultivation in liquid Dubo's medium when PL and products of their hydrolysis (lyso-components) in various concentrations were added (table 1). PL were incorporated in the cultivation medium in the form of large unilamellar vesicles. The cells were grown in atomized system BACTEC, in which the growth is detected due to the alterations in oxygen uptake level by means of fluorescent indicator, that is quenched by high oxygen concentrations (fig. 3).

Fig. 3. Growth curves of *M. tuberculosis* H37Rv in the presence of PL liposomes obtained by means of BACTEС. 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml). Reproduced from [Andreevskaya, 2010]

Viability of the cells was observed using colony counting on solid medium (Dubo's agar) after isolation of the passage that was exposed to the lipids in the liquid medium (fig. 4). It was found that addition of negatively-charged CL into cultivation medium had an effect on the growth rate and viability of *M. tuberculosis* H37Rv, while electro-neutral PC wasn't active. As it follows from the data shown in table 1 the effect of CL was dose-dependent: low concentration (50 µg/ml) caused the 1-2 day delay in growth of mycobacterial cells, and 250-330 µg/ml concentration fully inhibited growth. Applying Murohashi staining method we demonstrated that such concentration of CL cause lysis of *M. tuberculosis* H37Rv cells (fig. 5) [Sorokoumova, 2009].

Fig. 4. A photography of macrocolonies of *M. tuberculosis* H37Rv on Dubo's agar by the 18th day of cultivation. 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml). Reproduced from [Sorokoumova, 2009]

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 245

Fig. 5. Murohashi staining of *M. tuberculosis* cells (14th day of growth). 1-H37Rv;

number [Sorokoumova, 2009] (fig. 6).

Reproduced from [Sorokoumova G.M., 2009]

2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml). Reproduced from [Mikulovish, 2010]

Fig. 6. Growth curves of *M. tuberculosis* H37Rv in Dubo's medium in the presence of CL liposomes. Growth was detected with real time PCR. 1-H37Rv; 2-H37Rv+CL (50 µg/ml);

CL is an instable compound that transforms in several substances in water phase. Both in Dubo's medium, containing high concentration of iron ions, and in Tris-buffer, which doesn't have an iron, the products of hydrolysis were mainly formed: lyso-, bislysocomponents and FA (fig. 7). Along with this components such products of

3-H37Rv+CL (250 µg/ml) ; 4-H37Rv+CL (500 µg/ml); 5-H37Rv+CL (750 µg/ml).

These data correlate well with the results of mycobacterial DNA detection by real time PCR, according to which appearance CL in culture medium didn't lead to increase of DNA


Table 1. An effect of different concentrations of PL and the products of their hydrolysis on the start and intensity of *M. tuberculosis* H37Rv growth in Dubo's medium registered with BACTEC MGIT960.

250 16-18 + (considerably lower

lipid added The start of

growth, days

An absence of growth (-) µg/ml µM

more - -

A presence of growth (+)

than control)

Concentration of the

1 Control - - 7-8 + 2 CL 50 35 8-9 + 125 90 8-9 +

3 LisoCL 35 7-8 + 200 7-8 +

4. Bisliso-CL 35 7-8 + 200 7-8 +

5 Linoleic acid 10 7-8 + 90 7-8 + 200 14-15 +

 600 - - 2 Phosphatidic acid 50 8-9 + 335 - - 502 - - 3. Phosphatidylglycerol 50 8-9 + 335 - - 502 - -

glycerol 50 6-7 + 335 2-7 + 502-600 2-7 +

glycerol + linoleic acid 50 5-7 + 335 1-4, 8-9 + 502 1-4 - 670 - - 6 PC 500 - + 7 LisoPC 90 7-8 + 900 16 + 9000 16 +

(1:1, mole), 90 7-8 +

Table 1. An effect of different concentrations of PL and the products of their hydrolysis on the start and intensity of *M. tuberculosis* H37Rv growth in Dubo's medium registered with

400 20 + (slight growth)

300 – 700 200 and
