Determination of minimum inhibitory concentration (MIC) of some 1,2,4-triazole derivatives with potential tuberculostatic and tuberculocidal ability in vitro

The problem of tuberculosis infection caused by multidrug-resistant strains is becoming increasingly important in the world. This infectious disease poses a global health risk with an incidence rate of 8.8 million cases and a fatal outcome of 1.4 million. Coinfection with human immunodeficiency virus (HIV) increases the number of cases of tuberculosis and the development of active tuberculosis. Therefore, the search for substances with potential anti-TB activity is a promising way to solve this problem. 1,2,4-triazole derivatives – one of the interesting classes of anti-TB compounds. The study of their tuberculocidal, tuberculostatic properties, as well as MIC (minimum inhibitory concentration) was promising in relation to mycobacteria of different species. The work was conducted in 2019–2020 at Dnipro State Agrarian and Economic University. M. bovis and M. fortuitum mycobacterial species were selected for the study. The investigation of MIC levels for isoniazid, GKP-305, BKP-100 revealed that the growth of M. fortuitum had begun at 12th and 14th day on Sauton and Model media modified with 0.125% hydrogumate solution, respectively, whereas growth on pure Sauton and Model media was observed on the 25th and 28th day, respectively. It indicates that modification of Sauton and Model media with 0.125% hydrogumate solution doubles growth rate of M. fortuitum. As for isoniazid added to Sauton medium, MIC in case of M. fortuitum was impossible to determine since the growth was observed even at 50 μg/mL. With respect to GKP-305 added to Sauton medium, MIC was 50 μg/mL, which was concluded by the absence of growth. However, in the range of 25 to 0.19 μg/mL, isolated flakes of the colony with matte finish up to strong, noticeable strokes of the colonies with matte finish, which become well apparent at shaking, were observed. Having conducted studies on MIC determination on M. bovis and M. fortuitum using various growth media, it was established that MIC for isoniazid, GKP-305, BKP-100 applied on M. fortuitum in Sauton medium was 50 μg/mL; on Model medium, only MIC for BKP100 on M. fortuitum could be determined, which was 50 μg/mL; similar results were obtained for 0.125% hydrogumate-modified Sauton medium and 0.125% hydrogumate-modified Model medium (MIC for BKP-100 was 50 μg/mL). The studies revealed the potential teberculocidal and tuberculostatic ability of the studied drugs GKP-305 and BKP-100 in relation to M. fortuitum and M. bovis. Isoniazid was used as a control. High efficacy was found in BKP-100 in relation to mycobacteria (MIC from 12.5 to 50 μg /mL). GKP-305 showed a tuberculostatic effect.


Introduction
The tuberculosis infection, which is caused by multidrug-resistant strains of bacteria, is a currently a growing global concern. (Zazharskyi et al., 2019;Palchykov et al., 2019). K. A. Abrahams et al. (2012) have reported that Gram-positive rod-shaped representatives of Mycobacterium tuberculosis (M. tuberculosis) is one of the main human pathogens, which causes tuberculosis (TB). This infectious disease poses a global health threat with 8.8 million yearly cases, 1.4 million of which are lethal. Coinfection with human immunodeficiency virus (HIV) increases frequency of TB and facilitates the development of active tuberculosis. Hence, searching for substances with potential anti-TB activity may pave the way for solving this problem. Derivatives of 1,2,4-triazole represent a group of chemicals with interesting properties, including antitubercular activity. Specifically, the promising tuberculocidic and tuberculostatic properties have been demonstrated, including also minimum inhibitory concentration (MIC) against various mycobacterial species (Zazharskyi et al., 2019;Palchykov et al., 2020).
Ukrainian Journal of Ecology, 10(6), 2020 In their study, N. Rastogi et al. (2000) have compared the activity of rifapentine and its metabolite (25-O-desacetylrifapentine) in vitro and rifampicin and rifabutin against M. tuberculosis, M. africanum, M. bovis і M. Bovis in BCG. MIC was determined radiometrically using Middlebrook 7H11 agar. The bacteriocidic activity was evaluated in parallel and at selected concentrations.
Bactec MIC values for substance sensitive M. tuberculosis isolates using rifapentine and its 25-O-desacetylrifapentine were 0.03-0.06 mg/L and 0.125-0.25 mg/L, respectively. Similar MICs have been obtained for M. africanum (0.03-0.125 and 0.125-0.50 mg/L, respectively), and M. bovis (0.063-0.25 and 0.125-1.0 mg/L, respectively), although MIC for BC bovis was way lower (0.008-0.063 mg/L for rifapentine and 0.016-0.125 mg/L for its metabolite). MIC values were determined using 7H11 agar medium, and were one or two dilutions higher than those with the use of Bactec broth. As compared to rifampicin and rifabutin, the inhibitory activity of rifapentin against the substance sensitive isolates was close to the activity of rifabutin, while the inhibition caused by 25-O-desacetylrifapentine was similar to rifampicin. However, rifapentin has been shown to have slightly better bactericidic activity than rifabutin at the same concentration level. Clinical isolates of М. tuberculosis possessing high rifampicin resistance (MIC ≥ 32 mg/L) were also resistant to rifabutin, rifapentine, and 25-O-desacetylrifapentine, although MICs for rifabutin in this case were slightly lower than those for rifapentin (Rastogi, 2000). Studies by L. J. McGaw et al. (2008), Zazharskyi V. V. et al. (2019) have denoted that naphthoquinones and other substances showing antimycobaterial activity against tuberculosis mycobacteria were previously obtained from Euclea species. In their research, the authors have used several components extracted from Euclea natalensis and E. undulata, including organic leaf extracts, and evaluated the effect against the zoonosis-causing M. bovis. The group of the studied microorganisms contained М. bovis BCG and fast-growing species of M. smegmatis and M. fortuitum. Acetone extract of E. natalensis showed potent activity against M. bovis (MIC = 26 µg/mL). 7-Methyluglon derivative of naphtoquinone has been demonstrated to possess the most potent activity, reaching MIC of up to 1.55 µg/mL the pathogenic M. bovis. M. bovis BCG has been found less sensitive to the studied compounds, compared to the pathogenic strain. M. smegmatis appeared to be a better predictor for antimycobacterial activity against the pathogenic M. bovis and M. tuberculosis, while MIC values, obtained using M. fortuitum, correlated well with the values for M. bovis BCG. M. Tato et al. (2006) have reported the activity of linezolid studied on 55 tubercular mycobacteria, including one M. bovis and two multidrug-resistant isolates of M. bovis using standard 7H10 agar dilution and standard ESP Culture System II method. Both methods have yielded similar MIC90 values (minimum inhibitory concentration for 90% of species) at 0.5 mg/L. However, the former method afforded slightly lower MIC50 values (minimum inhibitory concentration for 50% of species), namely 0.25 mg/L, while the latter gave 0.5 mg/L. No difference was noticed between the sensitive and resistant isolates, including multidrug-resistant isolates of M. bovis with MIC range of 0.12-0.5 mg/L. The authors think that potential role of linezolide in TB-infected organisms requires additional evaluation in vivo. Gemechu A. et al. (2013) have denoted that crude 80% methanol extracts of C. aurea roots, O. basilicum seeds, and the leaves of A. abyssinica, C. macrostachyus, and E. camaldulensis possess antimycobacterial activity with MICs ranging from 6.25 to 100 µg/mL. MIC values for 80% methanol extracts of the components described in an order above was the following: 25-100 µg/mL and 12.5-75 µg/mL, 25-100 µg/mL and 25-50 µg/mL, 6.25-50 µg/mL and 12.5-50 µg/mL, 12.5-100 µg/mL and 18.25-50 µg/mL and 6.25-50 µg/mL and 12.5-50 µg/mL for M. tuberculosis and M. bovis, respectively. In their study, Ahmad Z. et al. (2012) have shown that MIC for 5-chloropyrazinamide against M. tuberculosis was between 12.5 and 25 µg/mL, while the serum inhibition titer ratio was 1 : 4. When analyzed under the same conditions, MIC for pyrazinamide exceeded 100 µg/mL, whereas mice's serum did not possess any inhibitory activity passing the dose of 300 mg/kg. 5-Chloropyrazinamide was well tolerated by both non-infected and infected mice at doses of up to 300 and 150 mg/kg, respectively. At the same time, both pure pyrazinamide and in combination showed its regular antimicrobial activity in mice infected with M. tuberculosis without demonstrating activity against M. bovis, whereas 5-chloropyrazinamide did not show any activity against both M. tuberculosis and M. bovis. Pyrazinamide, which is an analogue of nicotinamide, is a first-line anti-TB medication with a unique sterilizing effect. The introduction of pyrazinamide into a combined TB treatment, which includes isoniazid, rifampin, and ethambutol, has been demonstrated to shorten treatment duration and, thus, enabled short-term TB treatment. Pyrazinamide is a prodrug, which is hydrolyzed by mycobacterial pyrazinamidase (nicotinamidaze) into pyrazinoic acid. Pyrazinoic acid is effective against M. tuberculosis, although its precise bacteriocidic mechanism of action is yet to be determined, It has been proposed that pyrazinoic acid disrupts membrane transport and energy balance in M. tuberculosis, or inhibits fatty acid synthetase I (FAS I). Pyrazinamidase is coded by the pncA gene, and mutations in pncA, which leads to the suppression of activity of amidases, enable resistance development to pyrazinamide in M. tuberculosis strains. Pyrazinamidesensitive and resistant isolates are usually sensitive to pyrazinoic acid in vitro, although pyrazinoic acid is inactive in vivo. It has been established that pyrazinoic acid esters and 5-substituted pyrazinoic acid derivatives are active against pyrazinamidesensitive and pyrazinamide-resistant M. tuberculosis strains, as well as against naturally pyrazinamide-resistant M. bovis, M. kansasii, and М. avium isolates, in vitro. 5-Chloropyrazinamide is one of such derivatives, which has also been shown to inhibit the growth of M. smegmatis at MIC of 25 µg/mL, which is way lower than pyrazinamide (4000 µg/mL). Both pyrazinamide and 5-chloropyrazinamide, along with their acidic form 5-chloropyrazinoic acid, have been investigated for in vitro activities against M. tuberculosis, M. bovis (which is initially resistant to pyrazinamide due to nicotinamidase deficiency) and a range of non-tubercular mycobacteria using broth dilution method. 5-Chloropyrazinamide demonstrates higher activity than pyrazinamide against all of the tested microorganisms. At neutral pH, MICs for pyrazinamide and 5-chloropyrazinamide against M. tuberculosis range between 32 and 2048 µg/mL and between 8 and 32 µg/mL, respectively. MICs for pyrazinoic acid and 5-chloropyrazinoic acid fluctuate between 16 and 64 µg/mL and between 64 and 256 µg/mL, respectively. Therefore, MIC values for 5-chloropyrazinamide and pyrazinoic acid show that M. tuberculosis is more sensitive these substances than to pyrazinamide and 5-chloropyrazinoic acid. Apart from that, pyrazinamide-resistant isolates are still sensitive 147 Determination of minimum inhibitory concentration (MIC) Ukrainian Journal of Ecology, 10(6), 2020 to 5-chloropyrazinamide, pyrazinoic acid, and 5-chloropyrazinoic acid in vitro, which illustrates that 5-chloropyrazinamide does not require the activation of the mycobacterial pyrazinamidaze. It has also been confirmed by the observation that 5chloropyrazinamide, unlike pyrazinamide, is active against M. bovis with MIC of 8 µg/mL in vitro and, unlike pyrazinamide, shows activity even at neutral pH. N. K. Taneja, J. S. Tyagi (2007) have determined MICs for a range of drugs against M. tuberculosis and BCG M. bovis. They used resazurin microtiter assay analysis to test the substances. The obtained results were in good correlation with CFU-based results. Metronidazole and nitrofurans showed mild bactericidal activity using hypoxic resazurin reduction assay. It has been highlighted that hypoxic resazurin reduction assay, unlike conventional CFU count, allows to distinguish between metabolically active and inactive bacteria. Hypoxic resazurin reduction assay assay showed good correctness using both fluorometric and visual approaches to distinguish between bacteriocidic and bacteriostatic effects of a given substance. The aim of the work is to determination of minimum inhibitory concentration (MIC) of some 1,2,4-triazole derivatives with potential tuberculostatic and tuberculocidal ability in vitro.

Material and methods
The work was conducted in 2019-2020 at Dnipro State Agrarian and Economic University. Substances GKP-305 and BKP-100 were synthesized according to the reported procedures at Zaporizhia State Medical University, Zaporizhia, Ukraine (O. А. Bigdan, A. S. Hotsulia). Synthetic studies were performed using reagents from Merck (Darmstadt, Germany) and Sigma-Aldrich (Missouri, USA). The structure and composition of the key synthesized compounds were confirmed by elemental analysis, NMR spectroscopy and chromato-mass spectrometry.
Mycobacterium strain M. bovis 100 passage, which was obtained from biological material of cattle (lymph nodes), isolated and typed by the tuberculosis Department of the regional laboratory of the state food and consumer Service of the Dnipropetrovsk region and M. fortuitum Museum strain ATCC 6841, deposited in the State scientific control Institute of biotechnology and microorganism strains.
M. bovis and M. fortuitum mycobacterial species were selected for the study. A broth used as a liquid growth medium for culturing was prepared at a dilution of 1 mg of bacterial culture per 1 mL of normal saline. The broth was prepared in aseptic conditions of the box using sterile porcelain mortar and pestle with further inoculation into liquid growth media prepared for culturing as specified (Model and Sauton medium) and, respectively, in modifications of these media, namely the combinations with 0.125% hydrogumate solution and fulvic acids solution. Model medium was prepared as follows: 20 mL of the medium were weighed into 40 test tubes on analytical balance AXIS AN 200 with the addition of potassium hydrogenphosphate -1.0 g; ammonium oxalate -1.0 g; iron (II) sulphate -0.01 g; magnesium sulfate -0.1 g; glycerol, pure -10 cm 3 ; and diluted with Aque purificateae to 200 cm 3 . Sauton medium was prepared as follows: L-asparagine -0.8 g; citrate, pure -0.4 g; potassium hydrogenphosphate -0.1 g; magnesium sulfate -0.1 g; iron ammonium citrate -0.01 g; and diluted with Aque purificateae to 200 cm 3 . The media were placed into 250 mL flasks, and then autoclaved during 30 min at 1,5 atm. Modified Model and Sauton media were prepared by adding 20 mL of 0.125% hydrogumate solution and 20 mL of 0.125% fulvic acids solution to the corresponding flasks (Zazharskyi et al., 2019;2019b;Palchykov et al., 2019a;Palchykov et al., 2020b).

Results
As a result of MIC studies for isoniazid, GKP-305, BKP-100, it was found that the growth of M. fortuitum culture began on days 12 and 14, respectively, on Sauton and Model in combination with 0.125% solution of humate, and on Sauton and Modelrespectively on the 25th and 28th day. It can be concluded that the modification of Sauton and Model media with 0.125% hydrogumate solution allows to 2 times accelerate the growth of colonies of M. fortuitum. The MIC was determined for isoniazid on Sauton medium against M. fortuitum as absent because growth was observed even in vitro with a concentration of 50 μg/mL. The MIC was determined for GKP-305 against M. fortuitum on Sauton medium as 50 μg/mL in the absence of colony growth.
Already in the following tubes from 25 to 0.19 μg/mL was characterized by growth from single flakes of matte color to intensive growth with braids and plaits of matte color, which are clearly visible when shaking (Fig. 1).
The growth was characterized from single flakes of matte color to intensive growth with braids and plaits of matte color, which are clearly visible when shaken in the following test tubes (from 12.5 to 0.19 μg/mL) (Fig. 2). Accordingly, in the control there was an intensive growth with the formation of braids, plaits and matte flakes (Fig. 2). The MIC for the M. fortuitum was defined for isoniazid on Model medium as absent because in vitro, even at a concentration of 50 μg/mL, there was growth in the form of single flakes (Fig. 3). Tuberculocidal effect of GKP-305 on M. fortuitum on Model medium has not been established. The MIC is set as absent or exceeding 50 μg/mL -marked growth of the culture of M. fortuitum (Fig. 4). BKP-100 inhibited growth of M. fortuitum with an MIC value of 50 μg/mL. Subsequent dilutions of BKP-100 showed the appearance of single colonies (MIC < 25 μg/mL) (Fig. 5). The MIC was defined for isoniazid in relation to M. fortuitum as absent or exceeding 50 μg/mL on Sauton medium in combination with 0.125% hydrogumate solution (Fig. 6). MIC for the GKP-305 was established as absent or exceeding 50 μg/mL on the Sauton medium in combination with 0.125% solution of hydrogumate in relation to M. fortuitum (Fig. 7). The MIC for BKP-100 was set as 50 μg/mL on Sauton medium in combination with 0.125% hydrogumate solution against M. fortuitum (Fig. 8). The MIC for BKP-100 was set as 50 μg/mL on Sauton medium in combination with 0.125% hydrogumate solution against M. fortuitum (Fig. 9). The MIC for the drug GKP-305 was established as absent or exceeding 50 μg/mL on Model medium in combination with 0.125% solution of hydrogumate in relation to M. fortuitum (Fig. 10). The MIC for BKP-100 was set as 50 μg/mL on Model medium in combination with 0.125% solution of Hydrogumate against M. fortuitum (Fig. 11). Fig. 11. The growth M. fortuitum on the medium of Model + Hydrogumate: left test tube -with BKP-100 (МІС 50 μg/mL, no growth), test tube in the middle -without BKP-100 (growth in the form intense flakes), right test tube -with BKP-100 (6.25 μg/mL, growth in the form of small single flakes) The MIC was determined for isoniazid (50 μg/mL) on Sauton medium in relation to M. bovis. Colony growth was observed at the following concentrations of 25 and 12.5 μg/mL (Fig. 12). The MIC was determined to be absent for GKP-305 in relation to M. bovis because growth was observed even in vitro with a concentration of 50 μg/mL. Growth in test tubes was characterized by the formation of single flakes of matte color to intense growth with braids and plaits of matte color, which are clearly visible when shaken (Fig. 13). The MIC was set as 12.5 μg/mL for BKP-100 on Sauton medium in relation to M. bovis. The growth was present in subsequent concentrations (Fig. 14). The MIC was determined for isoniazid against M. bovis on Model medium as absent because in vitro, even at a concentration of 50 μg/mL, there was growth in the form of single flakes (Fig. 15). The MIC was found for GKP-305 against M. bovis on Model medium as absent or greater than 50 μg/mL because even in a test tube with a concentration of 50 μg/mL culture growth was observed (Fig. 16). The MIC was determined for BKP-100 on Model medium for M. bovis: single colonies were observed in 50 μg/mL in the next 25 μg/mL tube, and intensive growth was observed in the control (Fig. 17). The MIC was found for isoniazid on Sauton medium in combination with 0.125% Hydrogumate solution against M. bovis as absent or greater than 50 μg/mL (Fig. 18). The MIC was found for GKP-305 on Sauton medium in combination with a 0.125% solution of fulvic acids against M. bovis as absent or greater than 50 μg/mL (Fig. 19). The MIC was found for isoniazid against M. bovis on Model medium in combination with 0.125% Hydrogumate solution as absent or greater than 50 μg/mL (Fig. 20). The MIC was found for GKP-305 against M. bovis on Model medium in combination with 0.125% Hydrogumate solution as absent or greater than 50 μg/mL (Fig. 21). The MIC was found for GKP-305 against M. bovis on Sauton medium in combination with 0.125% Hydrogumate solution as absent or greater than 50 μg/mL (Fig. 22).

Determination of minimum inhibitory concentration (MIC)
Ukrainian Journal of Ecology, 10(6), 2020 The MIC was found for GKP-305 against M. bovis on Model medium in combination with a 0.125% solution of fulvic acids as absent or greater than 50 μg/mL (Fig. 23). If we compare the growth rate of M. bovis colonies on Sauton and Model media in the modification with solutions of hydrogumate and fulvic acids 0.125%, we noted that the growth of mycobacteria with fulvic acids is twice as intense as with hydrogumate (Table 1)  Note: "-" -no growth; "+" -single colonies; "++" -medium-intensity growth with the formation of braids and plaits; "+++" -high growth intensity with the formation of a large number of flakes and braids Note: "-"-no growth; "+"-single colonies; "++"-medium-intensity growth with the formation of braids and plaits; "+++"-high growth intensity with the formation of a large number of flakes and braids Discussion A. Khan et al. (2008) have described in vitro anti-TB potential of 2-nitromidazole. Minimum bactericidal concentrations (MBcCs) for the substance against M. bovis BCG and M. tuberculosis (H37Ra), which replicate actively, were determined to be at 0.226 µg/mL and 0.556 µg/mL, respectively. MICs were 100 times lower than previously reported values for nitroimidazole derivatives, such as nitrofurantoin and furaltadone, which highlights a considerable potential of the heterocycle. 2-Nitroimidazole did not pose any significant effect on saprophytes. It has also been revealed that the substance is active against M. tuberculosis, for isoniazid (fractional inhibitory concentration index (FICi) 0.125-0.375), rifampicin (FICi 0.118-0.281), and streptomycin (FICi 0.094-0.275), respectively. MICs for first-line medications in the presence of glycyrrhetinic acid-30-piperazine were 4-16-fold reduced: isoniazid (FICi 0.094-0.266), rifampicin (FICi 0.114-0.313), streptomycin (FICi 0.094-0.281). Moreover, MICs for 18-βglycyrrhetinic acid or glycyrrhetinic acid-30-piperazine only were significantly reduced 8 to 16-fold, 8 to 64-fold, and 8 to 128fold in the presence of isoniazid, rifampicin, and streptomycin, respectively. These results indicate that 18-β-glycyrrhetinic acid and its derivatives are promising antimycobacterial drugs. Arai et al. (2013) determined that biofilm produced by pathogenic bacteria protects them from antibiotics and immune system of the host. During the search for new inhibitors of biofilm formation in Mycobacterium species, the authors have highlighted terpenoids ophiobolin C, ophiobolin K, and ophiobolin G recovered from the culture of a sea fungus Emericella variecolor. The mentioned ophiobolins inhibit biofilm formation of M. smegmatis with MIC of 4.1-65 µM. The specified compounds did not show antimicrobial activity at concentrations at which anti-biofilm activity was observed. Ophiobolin K was also effective against biofilm formation in М. bovis BCG and enabled antimicrobial activity of isoniazid.
For both species, MIC for chloramphenicol was 5 µg/mL. Both species were inhibited by diacetyl chloramphenicol at the concentration of 50 µg/mL. However, it is possible that both cultures were actually sensitive to diacetyl chloramphenicol. Most probably, diacetyl chloramphenicol was converted into chloramphenicol, which inhibited the growth of the specified species. Shishido et al. (2007) have revealed that M. bovis bacille Calmette-Guérin (BCG) vaccine is the only viable vaccine against TB due to its highly valuable protective properties and low virulence. However, there were cases of systematic infection caused by this vaccine in individuals with weakened immunity. It is known that isoniazid, RMP, SM, and EMB are effective as anti-TB treatment, hence being used to treat BCG infections. Unfortunately, however, the evidence on sensitivity of vaccine strains of BCG to the mentioned substances is scarce. Pan et al. (2011) have denoted that there is a substantial demand for new anti-TB medications possessing new mechanisms of action due to growing concern of global TB spread. Having conducted the search for antimycobacterial metabolites among endophytes extracted from mangrove trees, the researchers have established that the active component of the extract from Fusarium sp. is fusaric acid. Antimycobacterial analyses have shown that its complexes with cadmium (II) and copper (II) possess a strong inhibitory activity against M. bovis BCG (MIC = 4 µg/mL) and H37Rv M. tuberculosis (MIC = 10 µg/mL) strains, respectively. It is one of the first reports on antimycobacterial activity of Fusarium metabolite and its metal complexes. Johar et al. (2005) have studied various 5-substituted 2'-desoxyuridines with respect to their inhibitory activity against M. bovis and M. avium. 5-(С1-substituted)-2'-desoxyuridine derivatives appeared to be potent inhibitors of M. avium (MIC90 = 1-5 µg/mL).
According to the researchers, the nature of the substituents at C-5 in 2'-desoxyuridine range, is the determinant factor of antimycobacterial activity. This new class of inhibitors is promising for research and development of new anti-TB medications. During the search for antimycobacterial substances, Arai et al. (2015) have highlighted nibomycin, which was identified in cultural broth of sea origin Streptomyces sp. The compound showed antimicrobial activity against M. smegmatis and M. bovis BCG with a MIC of 1.0 µg/mL, both in in active growth in aerobic conditions and in hypoxic conditions. The substance was also effective against M. tuberculosis, including clinical strains. The studies have revealed that the mechanism of nibomycin activity is linked with the interaction with enzymes, which unwind DNA helices before cell division. As a result, morphological alteration of mycobacterial cells occurs, leading to bacterial cell death. Fujiwara et al. (2018) have determined that delamanid, a nitrodihydroimidazooxazole derivative, is effective against M. tuberculosis. Its mechanism of action involves the disruption of cellular membrane components of M. tuberculosis (namely, mycolic acids). Delamanid is activated by F420 enzymes of the pathogen using nitroreductase Rv3547 as a prodrug. Sambhaji T. Dhumal et al. (2016) claim that new 2-pyridinesubstituted thiazolyl-5-aryl-1,3,4-oxadiazoles were synthesized in high yields and tested for anti-tuberculosis activity in vitro against M. tuberculosis H37Ra (MTB) and M. bovis BCG. Among them, a number of compounds showed promising activity against BCG M. bovis in concentrations less than 3 μg/mL. A molecular docking study was also performed on the enzyme mycobacterial enoyl reductase to understand how these molecules bind. The properties of ADME named products were also analyzed. Desai et al. (2016) have shown in their research that a series of 1,3,4-oxadiazole derivatives based on indole and pyridine synthesized and evaluated them in vitro antitubercular activity against Mycobacterium tuberculosis H37Ra and Mycobacterium bovis BCG in both active and dormant condition. The leading compound showed very good anti-TB activity. Zhao et al. (2016) have determined that cyadox, mecindox and quinocetone in combination with rifampicin have an additive effect on M. tuberculosis with a fractional inhibitory concentration (FIC) index of 0.75.
The problem of tuberculosis infection caused by multidrug-resistant strains is becoming increasingly important in the world. Abrahams et al. (2012) advise that gram-positive bacillus Mycobacterium tuberculosis -is the main human pathogen and the causative agent of tuberculosis (TB). This infectious disease poses a global health risk with an incidence rate of 8.8 million cases and a fatal outcome of 1.4 million. Coinfection with human immunodeficiency virus (HIV) increases the number of cases of tuberculosis and the development of active tuberculosis. Therefore, the search for substances with potential anti-TB activity is a promising way to solve this problem. 1,2,4-triazole derivatives -one of the interesting classes of anti-TB compounds. The study of their tuberculocidal, tuberculostatic properties, as well as MIC (minimum inhibitory concentration) was promising in relation to mycobacteria of different species.
Ukrainian Journal of Ecology, 10 (6), 2020 The studies revealed the potential teberculocidal and tuberculostatic ability of the studied drugs GKP-305 and BKP-100 in relation to M. fortuitum and M. bovis. Isoniazid was used as a control. High efficacy was found in BKP-100 in relation to mycobacteria (MIC from 12.5 to 50 μg /mL). GKP-305 showed a tuberculostatic effect.

Conclusion
The investigation of MIC levels for isoniazid, GKP-305, BKP-100 revealed that the growth of M. fortuitum had begun at 12th and 14th day on Sauton and Model media modified with 0.125% hydrogumate solution, respectively, whereas growth on pure Sauton and Model media was observed on the 25th and 28th day, respectively. It indicates that modification of Sauton and Model media with 0.125% hydrogumate solution doubles growth rate of M. fortuitum. As for isoniazid added to Sauton medium, MIC in case of M. fortuitum was impossible to determine since the growth was observed even at 50 µg/mL. With respect to GKP-305 added to Sauton medium, MIC was 50 µg/mL, which was concluded by the absence of growth. However, in the range of 25 to 0.19 µg/mL, isolated flakes of the colony with matte finish up to strong, noticeable strokes of the colonies with matte finish, which become well apparent at shaking, were observed.