V processe formirovaniya ustojchivosti M. tuberculosis k ftorhinolonam v organizme hozyaina mogut odnovremenno sosushchestvovat' puly M. tuberculosis, chuvstvitel'nye k preparatam etoj gruppy, i puly M. tuberculosis s razlichnymi determinantami ustojchivosti. Cel'yu issledovaniya bylo izuchit' osobennosti rosta in vitro shtammov M. tuberculosis, otlichayushchihsya geneticheskimi determinantami ustojchivosti k ftorhinolonam, v usloviyah konkurencii za pitatel'nye veshchestva. Issledovanie provedeno na pyati klinicheskih shtammah M. tuberculosis s mnozhestvennoj lekarstvennoj ustojchivost'yu, razlichayushchihsya strukturoj gyrA. SHtammy kul'tivirovali poparno i individual'no v optimal'nyh usloviyah (sreda Middlebrook 7H9) i v usloviyah mul'tistressa (50% sreda Middlebrook 7H9, 2 mM KNO2, 0,02% H2O2). Vremya eksperimenta sostavilo 21 sutki. CHislo kletok kazhdogo iz sovmestno kul'tiviruemyh shtammov ocenivali po kalibrovochnym krivym zavisimosti porogovogo cikla polimeraznoj cepnoj reakcii po celevomu dlya mutacii kanalu ot koncentracii kletok M. tuberculosis. Po chislu kletok kazhdogo shtamma pri sovmestnom kul'tivirovanii vychislyali velichinu konkurentnogo fitnesa i udel'nuyu skorost' rosta. Bylo ustanovleno, chto shtammy M. tuberculosis s mutaciyami v gyrA ustupali v skorosti rosta shtammu s dikim tipom gyrA, chto bylo osobenno sil'no vyrazheno v usloviyah mul'tistressa. SHtamm s naibolee rasprostranennoj mutaciej gyrA_D94G imel naimen'shuyu skorost' rosta iz vsekh issledovannyh shtammov. Byla vyskazana gipoteza, chto medlennyj rost M. tuberculosis s etoj mutaciej mozhet privodit' k tolerantnosti k protivotuberkuleznym preparatam i v rezul'tate etogo shtamm poluchaet preimushchestvo v usloviyah himioterapii po sravneniyu s drugimi mutantnymi po gyrA variantami.

As M. tuberculosis strains develop resistance to fluoroquinolones, pools of M. tuberculosis sensitive to drugs of this group and pools of M. tuberculosis with different resistance determinants can simultaneously coexist in the host organism. The goal of this research was to run an in vitro investigation of growth characteristics of M. tuberculosis strains which have different genetic determinants of resistance to fluoroquinolones, in the setting of competition for nutrients. The research used five clinical strains of multidrug-resistant M. tuberculosis differing in gyrA structure. Strains were cultured in pairs and individually under optimal conditions (Middlebrook 7H9 medium) and under conditions of multistress (50% Middlebrook 7H9 medium, 2 mM KNO2, 0.02% H2O2). The experiment took 21 days. The number of cells of each co-cultured strain was estimated from calibration curves. These curves showed the dependence of the threshold cycle of the polymerase chain reaction — respective to the channel targeted by the mutation — on the concentration of M. tuberculosis cells. The competitive fitness value and specific growth rate were calculated from the number of cells of each strain when co-cultured. M. tuberculosis strains with mutations in gyrA were found to be inferior in growth rate to the wild-type gyrA strain, which was particularly pronounced under multistress conditions. The strain with the most common gyrA_D94G mutation had the lowest growth rate of all strains examined. It has been hypothesised that the slow growth of M. tuberculosis with this mutation may lead to tolerance to anti-tuberculosis drugs, and as a result, the strain gains an advantage under chemotherapy conditions compared to other gyrA mutant variants.

The diagnosis of mycobacterial co-infection is one of the pressing public health issues. The study was aimed to determine discriminatory power of multiplex PCR used for species identification when detecting mixed mycobacterial populations. The study involved model samples representing the mixtures of DNA of two mycobacterial species with the ratios of 1 : 1, 1 : 9, 1 : 99, and 1 : 999 and different total DNA concentrations (103 gEq/mL to 106 gEq/mL). The model samples were assessed using the multiplex PCR-based AmpliTube-RV-Differentiation kit (Syntol LLC; Russia). It has been shown that the kit is capable of detecting the mixtures of mycobacterial species with high discriminatory power. The discriminatory power of real-time PCR used for analysis of the mixture of DNA of two mycobacterial species depended on the total DNA content in the sample and varied between 0.1% for high-rate samples (total DNA concentration 106 gEq/mL) and 50% for low-rate samples (total DNA concentration 103 gEq/mL) and corresponded to the amount of DNA of the species in the sample of at least 5 × 102 gEq/mL. When the amount of DNA of each species in the mixture was at least 5 × 102 gEq/mL, the results of PCR test for detection of co-infection did not depend on the mucobacterial species contained in the mixture, which should be taken into account when analyzing PCR results.

Diagnostika mikobakterial'noj koinfekcii — odna iz aktual'nyh problem zdravoohraneniya. Cel'yu issledovaniya bylo opredelit' diskriminiruyushchuyu sposobnost' metoda mul'tipleksnoj PCR vidovoj identifikacii pri vyyavlenii smeshannyh populyacij mikobakterij. Issledovanie vypolneno na model'nyh obrazcah, predstavlyayushchih soboj smes' DNK mikobakterij dvuh vidov v sootnoshenii 1 : 1, 1 : 9, 1 : 99 i 1 : 999 s raznoj summarnoj koncentraciej DNK (ot 103 GE/ml do 106 GE/ml). Model'nye obrazcy issledovali naborom «Amplitub-RV-differenciaciya» («Sintol»; Rossiya), osnovannom na mul'tipleksnoj PCR. Pokazano, chto nabor sposoben vyyavlyat' smesi vidov mikobakterij s vysokoj diskriminiruyushchej sposobnost'yu. Diskriminiruyushchaya sposobnost' metoda PCR v rezhime real'nogo vremeni pri analize smesi DNK dvuh vidov mikobakterij zavisela ot summarnogo soderzhaniya DNK v obrazce i var'irovala ot 0,1% dlya vysokonagruzhennyh obrazcov (summarnaya koncentraciya DNK × 106 GE/ml) do 50% dlya nizkonagruzhennyh obrazcov (summarnaya koncentraciya DNK × 103 GE/ml) i sootvetstvovala kolichestvu DNK vida v smesi ne menee 5 × 102 GE/ml. Pri kolichestve DNK kazhdogo vida v smesi ne menee 5 × 102 GE/ml rezul'tat PCR na vyyavlenie koinfekcii ne zavisel ot vida mikobakterij, vhodyashchih v smes', chto neobhodimo uchityvat' pri analize rezul'tatov PCR.

The purpose of the present study was to evaluate a real-time PCR system for 12 nontuberculous mycobacteria (NTM) species identification developed by Central Tuberculosis Research Institute (CTRI; Moscow, Russia) in cooperation with Syntol LLC (Moscow, Russia). NTM cultures (210 strains, 19 species), Mycobacterium tuberculosis complex (MTBC) cultures (21 strains, 2 species), non-mycobacterial microorganisms (18 strains, 13 species) were used for the first stage of the assay evaluation. Clinical samples (sputum, N = 973) positive for smear microscopy and MTBC/NTM DNA by a PCR-based screening assay collected from 819 patients were used for specificity and sensitivity evaluation. Sensitivity for determining the NTM species directly from diagnostic material was 99.71%, with the specificity of 100%. The sensitivity and specificity for NTM species identification in cultures was 99.67% and 100%, respectively. Both sensitivity and specificity for determining MTBC in cultures was 100%.

AbstractTranscription activator‐like effectors (TALEs), which induce the expression of specific plant genes to promote infection, are the main pathogenic determinants of various Xanthomonas bacteria. However, investigation of TALEs from Xanthomonas campestris pv. campestris, which causes black rot disease of crucifers, received little attention. In this study, we used PCR‐based amplification followed by SMRT amplicon sequencing to identify TALE genes in several X. campestris pv. campestris strains. Computational prediction in conjunction with quantitative reverse transcription PCR analysis was used to find their targets in the Brassica oleracea genome. Transcription factor ERF121, from the AP2/ERF family, was identified as target gene for the conserved TALEs from multiple X. campestris pv. campestris strains. Several members of this family from diverse plants were previously identified as targets of TALEs from different Xanthomonas species. We propose that TALE‐dependent activation of AP2/ERF transcription factors promotes susceptibility to Xanthomonas through the misregulation of plant defence pathways.

Summary - TAL effectors (TALEs), which induce the expression of specific plant genes to promote infection, are the main pathogenic determinants of different Xanthomonas bacteria. However, investigation of TALEs from Xanthomonas campestris pv. campestris , which causes black rot disease of crucifers, is in its infancy. - In this study, we used PCR-based amplification in conjunction with SMRT amplicon sequencing to identify TALE genes in several Xanthomonas campestris pv. campestris strains and performed computational prediction in conjunction with RT-PCR-based analysis to identify their target genes in Brassica oleracea . - Transcription factor from the AP2/ERF family was predicted to be putative target gene for the conserved TALEs present in multiple Xanthomonas campestris pv. campestris strains. Its expression dramatically increased upon leaf inoculation with strains harbouring such TALEs. - Several members of the AP2/ERF factor family from different plant species were identified as targets of TALEs from various Xanthomonas species, which suggests that they constitute a new pathogenicity hub in plant-Xanthomonas interactions.

A new presumably simple consortium of a Leptolyngbya sp. and a Porphyrobacter sp. was isolated from Tolbo Lake in Mongolia. The draft genome sequences of both species are reported.

43 65.66 Таблица. Характеристика полученных геномных сборокTable.Characteristics of obtained genomic assembles

ABSTRACT We report here two draft cyanobacterial genome sequences, those of Cyanobacterium aponinum IPPAS B-1201, isolated from a hot spring in the Turgen Gorge (Kazakhstan), and the uncharacterized cyanobacterium IPPAS B - 1203, isolated from a hot spring in Karlovy Vary (Czech Republic). These two strains were deposited at the Collection of Microalgae (IPPAS) of the Timiryazev Institute of Plant Physiology.

The purpose of the present study was to create a real-time PCR test system allowing simultaneous detection of nontuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTBC) both in culture and sputum. NTM cultures (18 strains, 18 species), MTBC cultures (16 strains, 2 species) and non-mycobacterial microorganisms from the collection of the Central Research TB Institute (CTRI) were used for the preliminary evaluation of the test system. 301 NTM cultures from patients with mycobacteriosis were used to assess the sensitivity of the developed test system. Clinical respiratory samples (sputum) from 104 patients with mycobacteriosis, 3627 patients with tuberculosis and 118 patients with other lung diseases were used for diagnostic sensitivity and specificity testing. The specificity and sensitivity of the assay for MTBC was found to be 100% both in culture and sputum samples; for NTM, the specificity was 100% in culture and sputum, the sensitivity reached 100% in culture and 73.1% in sputum samples. Positive predictive value (PPV) and negative predictive value (NPV) of the assay for culture were both 100%, for clinical material 100% and 80.8%, respectively. The limit of detection at the probability of detection 95% (LoD95%) was estimated to be 16 cfu/ml for M. tuberculosis H37RV and 1200 cfu/ml for M. avium.

We report here the draft genome sequence of Mycobacterium heckeshornense, isolated from the sputum of a patient admitted to a tuberculosis hospital with suspected pulmonary tuberculosis.

Development of rapid detection of NTM in mycobacteriosis patients is critical. The aim of this study was the development of real-time PCR system, allowing simultaneous detection of MTB and NTM DNA in samples from patients with TB or mycobacteriosis. Materials and methods. NTM cultures were obtained from sputum using BACTEC MGIT 960. NTM species were identified using the Genotype CM/AS kits (HainLifescience, Germany). Primers and probes were designed using Primer 3 and Primer BLAST Software. 1007 NTM cultures isolated from patients admitted to the CTRI with presumed mycobacteriosis in 2013-2017 were analysed. For the MTB/NTM DNA detection system development were selected 18 rapid- and slowgrowing NTM species each of which was obtained from sputum of the same patient more than ones. Developed oligos were added to the PCR system “Amplitub-RV”(Syntol, Russia). The oligos detected all 18 species and did not interfere with MTB DNA amplification. The resulting multiplex PCR system was tested on 14 non-mycobacterium strains, 1007 NTM cultures, 124 sputum samples from 33 patients with established mycobacteriosis, and 3627 sputum samples from patients with verified TB and positive cultures obtained in Bactec MGIT 960. The specificity of NTM DNA detection was 100%, sensitivity of NTM DNA detection from cultures was 100% and from diagnostic samples – 66.7%. All 3627 samples with positive MTB cultures were PCR-positive for MTB DNA and PCR-negative for NTM DNA. Simultaneous detection of MTB and NTM DNA in samples allows to detect the pathogen within 3 hours, differentiating MTB from NTM; this accelerates diagnosing and enrolling in the adequate treatment regimen.

Goal 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. gordonae (99/727; 13.62%), M. intracellulare (68/727; 9.35%), M. lentiflavum and M. fortuitum (по 66/727; 9.08%), M. abscessus (49/727; 6.74%), M. kansasii (48/727; 6.60%), M. xenopi (29/727; 3.99%). Prevalence of non-tuberculosis mycobacteria is described in respect of the regions.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

Goal 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 manufactureru0027s 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. gordonae (99/727; 13.62%), M. intracellulare (68/727; 9.35%), M. lentiflavum and M. fortuitum (по 66/727; 9.08%), M. abscessus (49/727; 6.74%), M. kansasii (48/727; 6.60%), M. xenopi (29/727; 3.99%). Prevalence of non-tuberculosis mycobacteria is described in respect of the regions.

ABSTRACT 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.

The aim of the study was to assess the efficacy of a diagnostic panel based on next-generation sequencing (NGS) and developed by our team, to diagnose a wide range of socially significant hereditary degenerative diseases of the brain. Materials and Methods. Using the diagnostic target NGS panel (powered by Illumina MiSeq, USA), designed to sequence the encoding area of 300 genes related to neurodegenerative diseases manifesting with movement and cognitive disorders, we performed a mutation screening of the DNA of 32 patients with an otherwise obscure diagnosis. Results. The application of the original genetic panel revealed a number of rare hereditary neurodegenerative pathologies with mutations in genes for spinocerebellar ataxias and paraplegias, parkinsonism, dystonias, and neurometabolic diseases, identified by NGS and confirmed by direct sequencing. In 11 patients, 12 mutations were found in 10 different genes, causing the development of three autosomal recessive diseases (genes DDHD1, NPC1 and RARS ) and eight diseases associated with autosomal dominant inheritance (genes SPAST, SPTBN2, GRN, GCH1 , LRRK2, NOTCH3 and AGER ). Conclusion. Panel screening of uncertain cases of neurodegenerative diseases using this bespoke target model enabled us to reveal and subsequently confirm mutations in various genes for more than a quarter of the examined patients.

Goal of the study: to define the design of primers and probes specific to DNA of non-tuberculous mycobacteria and evaluate their diagnostic value in case of simultaneous detection of non-tuberculous mycobacteria and M. tuberculosis complex by real time PCR.Materials and methods. Primer 3, Primer BLAST, Ugene Uni Pro were used to design primers and probes. Preliminary assessment of specificity and sensitivity of detection of non-tuberculous mycobacteria DNA was performed on cultures belonging to 18 types of non-tuberculous mycobacteria, 16 strains of M. tuberculosis complex and 14 types of microorganisms being none Mycobacterum. Analytic sensitivity was tested on 284 cultures of non-tuberculous mycobacteria and diagnostic sensitivity was tested on 124 sputum samples. The kit ofM-Sorb-Tub-Avtomat (ZAO Sintol) was used for DNA isolation. Cultures were subcultured on the liquid medium of Middlebrook 7H9 in Bactec MGIT 960. Cultures were identified with the use of standard microbiological techniques. Analysis of DNA isolated from cultures was performed by the reagent kit of GenoTypeCM/AS (Hain Lifescience, Germany).Results. 100% specificity and sensitivity of PCR was demonstrated in mycobacterial cultures and 100% specificity and 69-70% sensitivity was demonstrated in diagnostic material analysis.