We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (approximately 85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively.

The WHO has declared tuberculosis (TB) a global health emergency. Therefore, there is an urgent need to discover and develop new anti–TB drugs. Here we report on a new category of 5–substituted pyrimidine nucleosides as potent inhibitors of Myco–bacterium tuberculosis growth in vitro. A series of 2ʹ–deoxy–, 3ʹ–azido–2ʹ,3ʹ–dideoxy–, and 3ʹ–amino–2ʹ,3ʹ–dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M. tuberculosis in vitro. 5–Dodecyloxymethyl–2ʹ–deoxyuridine was found to be a potent inhibitor of M. tuberculosis propagation in vitro. In contrast, monophosphates of the tested nucleosides were devoid of antimycobacterial activity. This new class of inhibitors seems to be a promising chemotherapeutic agent against TB and merits further studies.

The effects of liposomes containing phospholipid cardiolipin without antibiotic and loaded with levofloxacin on the growth of Mycobacterium tuberculosis with extensive drug resistance were studied in vitro. Liposomes consisting of cardiolipin alone in a concentration of 335 μM completely suppressed the growth of M. tuberculosis. In order to reduce the minimum inhibitory concentration of cardiolipin, complex liposome preparation consisting of phosphatidylcholin/cholesterol/cardiolipin and loaded with levofloxacin was prepared. Due to this, the cardiolipin concentration was reduced to 33.5 μM (50 μg/ml) and concentration of levofloxacin - to 2 μg/ml.

ФГБНУ «Центральный НИИ туберкулеза», Москва, РоссияЦель исследования: установить виды нетуберкулезных микобактерий (НТМ), выделяемых из дыхательных путей больных в различных регионах РФ, используя для идентификации НТМ ДНК-стрипы� MoniToring oF sPecies diVersiTY oF non-Tuberculosis MYcobacTeria in THe soMe russian regions using dna-sTriPs oF genoTYPe MYcobacTeriuM cM/as (Hain liFescience, gerManY) T. G. SMIRNOVА, S. N. АNDREEVSKАyA, E. E. LАRIONOVА, I. yU. АNDRIEVSKАyA, V. V. USTINOVА, L. N. CHERNOUSOVА central Tuberculosis research institute, Moscow, russiaGoal of study: to define species of non-tuberculosis mycobacteria, isolated from the respiratory tract of the patients from various Russian regions using DNA strips for identification of non-tuberculous mycobacteria� Materials and methods. 1204 cultures of non-tuberculosis mycobacteria isolated on solid and liquid media from 727 patients in whom tuberculosis/mycobacteriosis was suspected were examined from 2011 to February, 2017� Species of non-tuberculosis mycobacteria were identified using DNA-strips of Hain Lifescience (Germany) GenoType ® Mycobacterium CM/AS in compliance with manufacturer's instructions� results. 17 species of non-tuberculosis mycobacteria were identified (11 species belonged to the group of those slow-growing and 6 species belonged to fast growing non-tuberculosis mycobacteria)� Slow-growing mycobacteria prevailed (564/727, 77�58%) and M. avium was the most prevalent (210/727; 28�89%)� The following mycobacteria were found (in descending order): M.

The growth of M. tuberculosis H37RV in culture medium was studied after addition of liposomes from different lipids (phosphatidylcholine, cardiolipin, and glycosphyngolipids). Addition of phosphatidylcholine into culture medium did not modify the growth and multiplication of mycobacteria. Addition of glycosphyngolipids and their mixture with phosphatidylcholine partially inhibited the growth. Addition of cardiolipin inhibited the growth of mycobacteria and even suppressed it, depending on the dose. Presumably, high concentrations of cardiolipin added into the culture medium, can transfer the mycobacteria into an uncultivable state.

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011-2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.

Here, we report the first draft genome sequence of the clinically relevant species Mycobacterium gordonae. The clinical isolate Mycobacterium gordonae 14-8773 was obtained from the sputum of a patient with mycobacteriosis.

2Ф ГБН У «В сероссий ск ий Н И И сельскохозяйственной биотехнологии», М осква Цель исследования: определить дизайн праймеров и зондов, специфичных к Д Н К нетуберкулезных микобактерий (Н Т М Б), и оценить их диагностическую значимость при одновременном вы явлении Н Т М Б и M. tuberculosis comp1ex (М Б Т К) методом П Ц Р в режиме реального времени. Материалы и методы. Дизайн праймеров и зондов осущ ествляли с использованием ПО Prim er 3, Prim er BLAST, Ugene Uni Pro. П ред варительную оценку специфичности и чувствительности вы явления Д Н К Н Т М Б проводили на культурах, принадлежащ их к 18 видам Н Т М Б, 16 штаммам М Б Т К и 14 видам микроорганизмов, не относящ ихся к роду M ycobacterum. Аналитическую чувствительность оце нивали на 284 культурах Н Т М Б, диагностическую чувствительность-на 124 образцах мокроты. Выделение Д Н К проводили набором «М-Сорб-Туб-Автомат» (ЗАО «Синтол»). Культуры подвергали субкультивированию на жидкой среде Midd1ebrook 7H 9 в системе Bactec M G IT 960. И дентификацию культур проводили с использованием стандартных микробиологических методов. Анализ ДН К, выделенной из культур, вы полняли c помощью набора реагентов G enoTypeCM /A S (H ain Lifescience, Германия). Результаты. Показаны 100%-ные специфичность и чувствительность П Ц Р при работе с культурами микобактерий и 100%-ная специфич ность и 69,70%-ная чувствительность при анализе диагностического материала.