Nontuberculous mycobacteria (NTM) include representatives of the genus Mycobacterium, except the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium leprae [1]. It is accepted to divide NTM into slow-growing and fast-growing ones based on the growth rate. Some species included in these groups can cause tuberculosis-like diseases of the lung and other organs [2–5].

The NTM infections represent a challenge faced by public health systems all over the world due to growing incidence and the associated mortality [6]. More than 90% of all registered cases of pulmonary diseases caused by NTM (mycobacteriosis) result from getting infected with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus [7]. The diagnosis of the diseases caused by NTM is challenging due to symptoms and x-ray features similar to that of tuberculosis. Furthermore, NTM and MTBC show no differences when bacterioscopically examined. It is difficult to treat the diseases caused by NTM, since NTM show natural resistance to the majority of broadspectrum antibacterial drugs and anti-tuberculosis drugs [8, 9].

Considering the complexity of treating mycobacteriosis, it seems relevant to search for the drugs effective against NTM, for example, to study the activity of novel anti-tuberculosis drugs. One such drug is bedaquiline, which was approved by the U.S. FDA and included in the WHO list of three drugs for treatment of tuberculosis caused by a pathogen with multiple and pre-broad drug resistance after clinical trials [10].

The NTM susceptibility to bedaquiline that was described in a limited number of studies [11–14] can fail to reflect the biological specifics of the NTM isolates represented in Russia. Bedaquiline susceptibility of the NTM circulating in the Russian Federation has been studied in a small number of species, and it is necessary to accumulate data [15–17].

The study aimed to determine bedaquiline susceptibility of the main clinically significant NTM species that were common in Russia.

METHODS

The study involved NTM isolates obtained from the patients examined at the Central Tuberculosis Research Institute in 2011–2024 at admission (one NTM isolate was obtained from each patient). The patients examined were residents of the following federal districts of Russia: Central (including Moscow and the Moscow region), Northwestern, Southern, Volga, and Urals regions.

NTM were identified to species by multiplex PCR, as previously reported [18]. The most common NTM species causing mycobacteriosis were included in the study: M. avium, M.intracellulare, M. chimaera, M. kansasii, M. xenopi, M. abscessus.

Bedaquiline susceptibility was determined by microdilution in a 96-well plate using the bedaquiline concentrations within the range of 0.125–4.000 µL/mL [19]. The bedaquiline fumarate substance containing 82.72% of the active ingredient (Janssen Pharmaceutica NV, Belgium) was used. The drug sample was dissolved in the chemically pure dimethyl sulfoxide to reach the necessary drug concentration, and the drug activity was taken into account.

A single-cell suspension was prepared from the NTM culture, while controlling CFU values using the spectrophotometer Ultraspec 10 (Ultraspec, USA) [20]. The resulting suspension was used to prepare a working suspension in the Middlebrook 7H9 medium with the concentration of 5 × 10⁵ CFU/mL and sown onto a plate, 100 µL per well [21]. Plates were incubated at 37 °С in the microbiological incubator Binder (Binder, Germany). The culturing time was 14 days for slow-growing and 5 days for fast-growing NTM. The bedaquiline minimum inhibiting concentration (MIC) was determined in each test, which corresponded to the highest drug dilution inhibiting the culture growth. To characterize bedaquiline susceptibility of each studied NTM species, MIC₅₀ and MIC₉₀ were determined, i.e. the minimum bedaquiline concentrations, to which 50% and 90% of strains of each NTM species were susceptible [22].

RESULTS

In 2011–2024, a total of 345 NTM isolates were obtained: 289 slow-growing NTM species (M. avium, M. intracellulare, M. chimaera, M. kansasii, M. xenopi) and 56 fast-growing ones (M.abscessus). Bedaquiline MIC values for the studied NTM species are provided in table.

Such slow-growing NTM species, as M. avium, M. intracellulare, M. chimaera, M. kansasii, showed high susceptibility to bedaquiline: the bedaquiline MIC mode and MIC₅₀ for all species were <0.125 µg/mL, MIC₉₀ was between < 0.125 and 0.5 µg/mL, depending on the species. One more slow-growing NTM species, M. xenopi, showed low susceptibility to bedaquiline with MIC₅₀ of 4 µg/mL and MIC₉₀ > 4 µg/mL. The fast-growing NTM species showed moderate susceptibility to bedaquiline with MIC₅₀ and MIC₉₀ of 1 µg/mL and 2 µg/mL, respectively.

When analyzing the resulting MIC distribution for all the studied slow-growing NTM species, a bimodal distribution was obtained allowing us to roughly determine that the bedaquiline tentative epidemiological cut-off value (ECOFF) for slow-growing NTM species did not exceed 1 µg/mL (figure).

Based on this tentative ECOFF value it can be concluded that 208/210 (99.05%; 95% CI 96.59–99.74%) of the studied M. avium strains, 19/20 (95.00%; 95% CI 76.39–99.11) M.intracellulare strains, 24/24 (100%; 95% CI 86.20–100.00) M. chimaera strains, 27/27 (100%; 95% CI 87.54–100.00) M. kansasii strains are susceptible to bedaquiline. Among all the studied M. xenopi stains no strain was revealed susceptible to the proposed bedaquiline ECOFF.

Among fast-growing NTM species, bedaquiline susceptibility was determined for only one species, M. abscessus, MIC distribution was unimodal, due to which bedaquiline ECOFF for fast-growing species was not determined.

DISCUSSION

The NTM natural resistance to most antibacterial drugs makes the problem of finding new drugs effective against this group of mycobacteria relevant. The study presented provides the data on bedaquline susceptibility assessment for the main clinically significant NTM species. The bedaquline susceptibility distribution for each NTM species was unimodal, which suggested the species-specific susceptibility of the studied NTM species to this drug. M. kansasii and NTM of the M. avium complex (МАС) that includes M. avium, M. intracellulare, M. chimaera, turned out to be highly susceptible to bedaquline (MIC mode — <0.125 µg/mL). M. abscessus was less susceptible (MIC mode — 1 µg/mL), and the lowest susceptibility was reported for M. xenopii (MIC mode — 4 µg/mL).

The results obtained for the NTM species included in МАС and for M. kansasii matched the results obtained in a number of studies; these suggest that bedaquline shows high activity against these NTM species [12–14].

The data on bedaquline susceptibility of M. xenopi strains is insufficient. According to some reports, bedaquline susceptibility of one laboratory M. xenopi strain was > 2 µg/mL, which is consistent with our results [12]. Another study describing bedaquline susceptibility of the NTM circulating in China involved assessment of four clinical M. xenopi strains, the bedaquline MIC for which varied broadly, from 0.0016 µg/mL to 1 µg/mL, which is significantly lower compared to the values we have determined [14].

In a number of studies the bedaquline ECOFF value for slow-growing NTM of 1 µg/mL was proposed, which matched the bedaquline ECOFF value determined in our study [13, 23]. It is interesting to note that this ECOFF is higher, than the bedaquline ECOFF for M. tuberculosis equal to 0.125 µg/mL, which also demonstrates higher NTM resistance to antituberculosis drugs relative to the tuberculosis pathogen [19].

The bedaquline susceptibility of the fast-growing NTM species M. abscessus was studied earlier [13]. According to the results of this study, the bedaquline MIC for M. abscessus had a bimobal distribution with the MIC modes of 0.13 µg/mL and > 16 µg/mL; the bedaquline MIC₅₀ and MIC₉₀ were 0.13 µg/mL and >16 µg/mL, respectively. In our study involving 56 M. abscessus strains, unimodal MIC distribution with the mode of 1 µg/mL was reported, and MIC₉₀ was 2 µg/mL. The differences revealed can indicate unique characteristics of the M.abscessus populations distinguished in Russia (the study presented) and China [13].

CONCLUSIONS

The study presented provides important information about the antibacterial drug susceptibility of the main clinically significant NTM strains circulating in the Russian Federation. Based on the data obtained it has been found that bedaquline is highly effective against МАС being the main causative agents of mycobacteriosis in the world and against M. kansasii. Other NTM species turned out to be less susceptible to this drug. Further research is necessary to accumulate the data on bedaquline susceptibility of various NTM species and determine the correlation between NTM susceptibility in vitro and bedaquline clinical efficacy when used to treat mycobacteriosis.