Search

The objective: to study drug susceptibility of M. avium and M. intracellulare isolates received from patients with mycobacteriosis residing in the North Western Federal District using the Sensititre SLOMYCO panels.Subjects and Methods. From 2014 to 2020, 192 slow-growing MAC strains (164 – M. avium, 28 – M. intracellulare) obtained from HIV-negative patients with pulmonary mycobacteriosis were studied. Drug susceptibility testing of MAC strains was performed using the Sensititre SLOMYCO panel consisting of 13 antibacterial drugs.Results. Of the 13 antibacterial drugs, clarithromycin was also most effective against both M. avium (67.1%; 110/164) and M. intracellulare (60.7%; 17/28) (p > 0.05). The proportion of M. avium and M. intracellulare strains sensitive to linezolid was high and amounted to 57.3 and 53.5%, as well as to amikacin – 57.3 and 53.5% respectively.Of the four antibacterial drugs of the Sensititre SLOMYCO panel (clarithromycin, moxifloxacin, linezolid, and amikacin), for which critical concentrations (CC) were established, clarithromycin was the most effective against M. avium and M. intracellulare. Antibiotic-resistant M. avium isolates were more frequent in the group of previously treated patients.

The aim of the study was to determine the taxonomic status of a group consisting of atypical strains of Acinetobacter baumannii, outline relevant characteristics and methods necessary for their identification. There were examined 10 strains of A. baumannii (6 of them primary comprised) bearing similar profile of atypical features isolated from clinical samples (urine, sputum) in 2017–2019 at the Military Medical Academy. Сlinical strains of typical A. baumannii (n = 36), Acinetobacter nosocomialis (n = 14), Acinetobacter pittii (n = 9) and 1 strain of Acinetobacter calcoaceticus isolated from the external environment were used in comparative studies. Atypical strains had the characteristics of A. calcoaceticus — A. baumannii (ACB) complex bacteria and were identified as A. baumannii. The utilization of substrates as the only carbon source was studied on a dense synthetic medium added with 0.2 % substrate during incubation for 72 hours at 37°C. Carbohydrate oxidation coupled to acid formation was detected on the Hugh–Leifson medium by using a micromethod. Aromatic amino acid biotransformation was carried out in liquid and dense nutrient media assessed in chromogenic reaction. The rpoB gene was used for strain genetic characterization. Amplification of two 940 and 1210 base pair (bp)-long fragments from the rpoB gene was performed by the routine polymerase chain reaction using primers with previously described sequences. Amplification products were sequenced by Sanger using Big Dye Terminator v3.1 (Applied Biosystems, USA) and capillary electrophoresis on an automatic sequencer ABI PRISM 3130 (Applied Biosystems, USA), followed by using methods for determining the similarity levels of sequenced fragments with the rpoB gene sequences of the reference strain A. baumannii ATCC 17978 (GenBank accession no. CP053098.1). It was found that all strains belonging to atypical A. baumannii spp. had a specific set of features that distinguish them from typical strains of A. baumannii as well as other types of the ACB complex: detected biotransformation of L-tryptophan (via anthranilate pathway) and anthranilic acid under unambiguous lack of such signs in other bacteria; lack of utilized sodium hippurate and L-arabinose being unambiguously evident in other bacteria; lack of utilized L-tryptophan, putrescine, L-ornithine being utilized in the majority of strains of belonging to other bacterial species. Genetic analysis showed that the control strains of typical A. baumannii displayed 99.20–99.21% similarity within the sequenced fragments of the rpoB gene with those from the rpoB gene of the reference strain. All 10 strains of atypical A. baumannii had similar features (99.20–99.21%). At the same time, parameters of control strains from other bacterial species significantly differed: A. nosocomialis (95.10–95.97%), A. pittii (94.63–94.92%), A. calcoaceticus (93.00%). Hence, the strains of atypical and typical A. baumannii are genetically homogeneous and belong to the same species. The data presented allow us to consider this group of atypical A. baumannii strains as a new biovar. We propose the name for this new biovar — tryptophandestruens (tryptophan-destroying) stemming from the Latin word destruens — destroying. Identification of A. baumannii bv. tryptophandestruens bacteria can be carried out in laboratory of any level by using tests for L-tryptophan biotransformation as well as sodium hippurate utilization.

The objective of the study: a comparative analysis of genome-wide sequences of seed lot (SL) 361 "sh" M. bovis BCG-1 (Russia) and its daughter isolates obtained from children with manifestations of BCG osteitis.Subjects and methods: genotyping; bioinformational analysis of genome-wide sequences of PS 361 "sh” vaccine BCG-1 (Russia) and its daughter isolates.Results. PS 361 "sh" BCG-1 (Russia) was heterogeneous in 7 coding sequences with different ratios of single nucleotide substitutions inherited by 2 of 6 daughter BCG isolates. At the same time, polymorphisms did not violate the genomic stability and viability of the BCG vaccine during long-term existence in vivo. In isolates of BCG 2925 and 5448, accumulation (hypothetically, under the pressure of selection in the vaccinated organism) of nonsynonymous substitutions in 4 out of 7 polymorphic genes was noted, including the genes ppsC, eccD5, and eccA5 associated with mycobacterial virulence. Evaluation of the significance of genomic variations in BCG isolates relating to the association with the development of post-vaccination osteitis requires more detailed information about the immune status of patients. Genome-wide analysis of production strains, seed lots, finished vaccine lots and daughter clinical isolates makes the contemporary approach to understanding the molecular basis of the efficacy and complications of BCG vaccination.

Among the members of the large group of non-tuberculous mycobacteria (comprising more than 180 species), M. avium subsp. hominissuis (MAH) is the most significant causative agent of pulmonary infection in immunocompetent individuals as well as disseminated infection in immunocompromised hosts, e.g. human immunodeficiency virus (HIV)-positive patients. Due to increased incidence rate of mycobacteriosis, especially in HIV infection, much still need to be learnt about the MAH genetic control and virulence mechanisms. Deciphering the genome contents of the M. avium strain 104 (isolated from an AIDS patient with disseminated MAH disease) allowed to compare genome sequences of M. avium strains to gain insights into genomic diversity associated with variable hosts and environments. Comparative genome analysis of MAH strains isolated from patients with pulmonary and disseminated forms of mycobacteri-osis revealed differences in the structure of the genome, affecting the key virulence genes. This review provides current data on the genetic determinants of MAH virulence associated with the initial phase of infection. Several mycobacterial virulence-associated gene families, such as mce (mammalian cell entry), mmp (mycobacterial membrane proteins), pe/ppe and esx expressed by MAH during human infection are thought to be crucial for adhesion, entry, survival, and reproduction inside host macrophages. The genetic mechanisms of MAH survival in human macrophage cell culture as well as mice exposed to toxic effects of reactive oxygen, nitric oxide, bactericidal proteins (cathelicidin) are discussed. The MAH survival in the latency-like state is important for pathogen dissemination. Some genetic and phenotypic features of MAH (absence of a cord factor, presence of plasmids, potential to “switch” morphological types of colonies) are compared with M. tuberculosis. In addition, we summarized current state of MAH drug discovery, a role of MAH intrinsic multidrug resistance, genetic control, as well as mechanisms underlying formation of resistance to various groups of antibiotics in MAH strains.

The objective of the study: to analyze the structure and trends in non-tuberculous mycobacteria (NTBM) population causing diseases in the North-Western Federal District of the Russian Federation.Subjects and methods. 745 clinical NTBM strains were identified. All clinical strains of NTBM were isolated by culture from clinical samples (sputum, bronchial washings, bronchoalveolar lavage fluid, pus, stool, urine, surgical and biopsy specimens, and venous blood in case of sepsis). In 23 cases, NTBM were detected post mortem. Mycobacteria were cultured on solid media or by the automated system of Bactec MGIT 960 BD USA. Species of re-cultured mycobacteria were identified using the test system of GenoType® Mycobacterium CM/AS (Hain Lifescience, Germany).Results. Analysis of the structure of the NTBM population isolated from patients with mycobacteriosis in the North-West of Russia, for the period of 2012-2018 showed overall dominance of M. avium (56.4%). However, certain NTBM species were found to be prevailing in some regions: M. lentiflavum and M. gordonae dominated in the Komi Republic and Arkhangelsk respectively. Unlike other regions of Russia, in the North-Western region, there was a consistently low level of detection of M. кansasii and M. xenopi. For the last 12 years in Saint Petersburg and Leningrad region, the variety NTBM in patients with different immune status has been growing but the portion of M. avium remains to be stably high exceeding 50%.

In the Kaliningrad region, the incidence of tuberculosis (TB) has declined significantly over the past decade (from 134/100 000 in 2006 to 50.6/100 000 in 2015), but still exceeds the average for the North-West Russia (40.7/100 000). In view of the increase in the proportion of primary multidrug resistance (MDR) of the causative agent from 23.9% in 2010 to 30.5% in 2015, the aim of this study was to analyze the current Mycobacterium tuberculosis population structure in the Kaliningrad region and to evaluate its spatial-temporal trends. The 73 M. tuberculosis isolates from patients with pulmonary TB newly diagnosed in 2015 were studied. Drug resistance (DR) was detected in 46 (63.0%) M. tuberculosis strains; of these, 32 (43.8%) were multidrug-resistant (MDR). The 46 (63.0%) of 73 M. tuberculosis strains were of the Beijing genotype with 19.2% (14 out of 73) share of the B0/W148 cluster. Spoligotyping of 27 non-Beijing M. tuberculosis strains made it possible to identify 15 spoligotypes of 5 genetic families — T, LAM, Ural, S, X. About half (16; 51.6%) of non-Beijing strains were represented by spoligotypes — SIT42, SIT53, SIT262, and SIT444. In a heterogeneous group T, mostly drugsusceptible strains of spoligotype SIT53 were prevailing. More than half (55.6%) of the LAM genotype strains exhibited DR. The Ural family was small (5.5%) and included strains of well-known in Russia M. tuberculosis spoligotypes SIT35 and SIT262, and one new, not found previously. MDR was statistically significantly associated with the Beijing genotype: 56.5% versus 25.9% in strains of other genotypes (in total) (P = 0.0134). Moreover, all 14 strains of epidemiologically and clinically significant B0/W148 cluster were MDR. The proportion of MDR strains of the Beijing genotype did not change significantly: in 2006 it was 61.3%, in 2015 — 56.5% (P = 0.6773). However, the spectrum of drug resistance to first-line anti-TB drugs expanded. If previously the majority (78.9%) of MDR strains additionally exhibited resistance to streptomycin only, in present study 72.0% of MDR strains were resistant to streptomycin, ethambutol, and pyrazinamide. The proportion of MDR strains of non-Beijing genotypes was 25.9%, a significantly higher than the similar indicators in 2006 — 2.2% (P = 0.0124). A relatively smaller proportion of strains of the Beijing genotype (50%) was found in TB patients from the shores of the Baltic Sea, a territory with a relatively more favorable socio-economic situation and a higher standard of living. Thus, the current epidemiological situation of TB in the Kaliningrad region is crucially determined by the growing circulation of MDR Beijing family strains, as well as a significant increase in the proportion of MDR strains of other genotypes.

Abstract. Mycobacteriosis is an infectious disease of animals and humans caused by non-tuberculosis mycobacteria including M. avium complex. Despite the fact that the transmission of M. avium from human to human has not been proved, and mycobacteriosis has been sporadic, the number of cases of disseminated forms of disease caused by M. avium among HIV-positive patients during the last ten years was increasing. Limited knowledge about the structure of M. avium population in Russia and the lack of simple methods for the microbiological diagnosis make difficult the epidemiological monitoring of mycobacteriosis. This facilitates the use of modern, efficient molecular genetic methods for the species and subspecies identification and typing of M. avium. Thus, the detection of mobile element IS901, restriction fragment polymorphism analysis of hsp65 gene and IS1245 allow the detection and subspecies identification of M. avium. The study of genomic polymorphisms of bacterial strains for the assessment of M. avium population structure became feasible due to a complex of molecular techniques: VNTR-typing, IS1245-and IS1311-RFLP-typing.