**2. Results**

### *2.1. Genetic and Phenotypic Characteristics of the Strain*

The Rostov strain of *Mycobacterium tuberculosis* was isolated in the South Federal District of Russia in 2013 from a patient with pulmonary tuberculosis.

The Beijing genotype (SIT1) was confirmed by spoligotyping. The assignment of the strain to a CAO clade of the Central Asian and Russian Beijing population (Beijing 94-32 cluster) was revealed by PCR assays as in [18,23], respectively. The next generation sequencing analysis on Ion Torrent additionally confirmed that the strain belongs to the CAO clade of the Beijing genotype and carries all previously described CAO-specific single nucleotide polymorphisms (SNPs) [21]. 24-locus MIRU-variable-number tandem-repeat (VNTR) typing scheme revealed 223325153533424682254423 profile which is designated 9358-25 in the international MIRU-VNTRplus database. This profile is closest to the 94-32 and has two di fferent loci, as shown in Figure S1.

According to the drug susceptibility testing and genome analysis, the strain belonged to pre-XDR tuberculosis and was resistant to streptomycin, isoniazid, rifampicin, ethambutol, kanamycin, amikacin, and capreomycin, as shown in Table 1. Additionally, a putative compensatory mutation in the *rpoC* gene (g764363a; G332S) was revealed.


**Table 1.** MIC distribution of the Rostov strain of *Mycobacterium tuberculosis* for antibiotics and drug resistance markers.

Note: MIC—minimal inhibitory concentration; R—resistance; S—sensitivity.

To compare the growth rate between the Rostov and H37Rv strains we determined a growth index and *C*max value, as shown in Figure 1. Growth index reflects that the Rostov strain grew faster than the H37Rv strain throughout the experimental period (*p* < 0.05), as shown in Figure 1A. The Rostov strain showed a higher *C*max than the H37Rv strain (*p* < 0.05), as shown in Figure 1B. *C*max for the Rostov strain was reached on the 25th day, whilst *C*max for the H37Rv strain was reached on the 30th day.

**Figure 1.** Growth of H37Rv and Rostov strains of *M. tuberculosis* in 7H9 broth. ( **A**)—Growth index (calculated by the colony-forming unit (CFU) at each time point divided by the CFU at initial time point); (**B**)—Comparison of *C*max (a maximum point on the growth curve); \*—values of *p* < 0.05.

### *2.2. Mice Survival Rate and Bodyweight Dynamic*

The model of *M. tuberculosis* infection of C57BL/6 mice was used for the comparison of the virulence of the Rostov clinical strain and the reference virulent strain H37Rv. Animals were intravenously injected with 5 × 10<sup>6</sup> CFU/mice of each strain (nine mice per strain, *n* = 18). Additionally, as a negative control, a group of uninfected animals (*n* = 9) was used. The patterns of animal survival were observed from the first to 90 days post-infection (p.i.). In each group of mice, weight control was performed. Figure 2 shows that mice infected with the Rostov and H37Rv strains started to die after 18 and 36 days of infection, respectively. All mice of the group infected with the Rostov strain succumbed to death within a 47-day period, while ~56% of mice infected by the H37Rv strain survived within a 90-day p.i. period. Mice weight analysis showed irreversible and severe depletion of animals infected with the clinical Rostov strain compared to animals infected with the laboratory H37Rv strain, as shown in Figure 3.

**Figure 2.** Comparison of the survival curves of C57BL/6 mice infected by *M. tuberculosis* strains. Data were analyzed by the Gehan–Breslow–Wilcoxon test. The value of *p* < 0.05 was taken as statistically significant.

**Figure 3.** Comparison of the weight changes curves of C57BL/6 mice infected by *M. tuberculosis* strains. The value of *p* < 0.05 was taken as statistically significant.

### *2.3. Investigation of Tuberculosis Process on the 30th Day of Infection*

To further define, the virulence of the studied strains, we investigated the tuberculosis process in the C57BL/6 mice models on the 30th day after pathogen injection, when all mice infected by the H37Rv strain were alive, and about 50% of mice infected by the Rostov strain were dead. The pathological processes provided by two *M. tuberculosis* strains were very different. Animal appearance after infection by the H37Rv strain was characterized by mild depletion and smooth fur, but after infection by the Rostov strain—by extremely emaciated and "ruffled" fur. The differences in survival times were associated with differences in the macroscopic appearance of lungs and liver harvested on the 30th day of infection, when more than 50% of mice infected with the Rostov strain were dead. It was shown that the lungs of mice infected by the Rostov strain were different from those in the H37Rv-infected mice, which appeared in increased lungs volume, intensively hyperemic, and no visible nodules; in turn, the lungs of mice infected by the H37Rv strain were pale pink colored with pale mass inclusions. The similar picture was obtained in the liver: Rostov-infected mice livers were dark brown with multiple nodules and the fatty degeneration was visible, while the livers from H37Rv-infected mice were smooth, dark brown, and normal volume, as shown in Table 2.


**Table 2.** Comparative characterization of the mortality, animal appearance and morphological description of internal organs of C57Bl/6 mice infected by the H37Rv and Rostov strains of *M. tuberculosis*.

The histological investigation of the C57Bl/6 mice infected intravenously by the H37Rv strain of *M. tuberculosis* at a dose of 5 × 10<sup>6</sup> CFU/animal showed a typical picture for TB mice models in the lungs: the small granulomas composed of numerous macrophages with abundant cytoplasm form; there are some lymphocytes between macrophages; dense perivascular lymphocytic infiltrates form in the lungs in addition to granulomas, as shown in Figure 4A. A single infiltrate consisting of few lymphocytes was found in histological sections of the liver, as shown in Figure 4C.

In contrast, when the C57Bl/6 mice were infected by the Rostov strain of *M. tuberculosis,* the pattern of pathological changes was different. Diffuse thickening of the alveolar septum due to an increased number of macrophages occurred in certain parts of the lungs. Lymphocytic infiltrates were not observed, as shown in Figure 4B. Microscopy of histological sections of the liver showed the presence of nodules consisting of focal cell accumulations in the parenchyma. Cellular infiltrates are composed of few typical macrophages and a large number of polymorphonuclear leukocytes, that indicate intensive pathogen multiplication and the increased development of a pathological inflammatory process, as shown in Figure 4D.

Bacterial load in the lungs and liver of mice infected with the Rostov and H37Rv strains was measured on day 30 p.i. According to the data presented in Figure 5, the clinical Rostov strain more actively proliferate in the parenchymatous organs of experimental animals than the H37Rv strains. The overall bacterial load in the lungs was higher than in the liver for both strains.

**Figure 4.** Histology of lungs and livers of C57Bl/6 mice on the 30th day after intravenous inoculation by the *M. tuberculosis* strains H37Rv (**A**—lungs, **C**—liver) and Rostov (**B**—lungs, **D**—liver). 1, 2, 3 and 4—×4, ×10, ×20 and ×40 magnification, respectively. The arrow indicates the specific mice cells (**A**4, **B**4—macrophages; **C**4—lymphocytes; **D**4—polymorphonuclear leukocytes).

**Figure 5.** *M. tuberculosis* cells loads of the C57Bl/6 mice lungs and liver on the 30th day after the inoculation of bacteria; \*—values of *p* ≤ 0.05; \*\*—values of *p* ≤ 0.01.
