Changes in Functional Activity of Neutrophils and Monocytes Isolated from the Peripheral Blood of Women at Different Phases of the Menstrual Cycle.

We studied functional activity of neutrophilic granulocytes and monocytes isolated from the peripheral blood of women during the follicular and luteal phases of the menstrual cycle. It was shown that phagocytic activity of neutrophilic granulocytes increases, their intracellular oxygen-dependent bactericidal activity decreases, and the number of monocyte extracellular traps increases in women in the luteal phase of the menstrual cycle in comparison with the follicular phase.

[The Spectrum of Mutations in Genes Associated with Resistance to Rifampicin, Isoniazid, and Fluoroquinolones in the Clinical Strains of M. tuberculosis Reflects the Transmissibility of Mutant Clones].

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.

Peculiarities of the Inflammatory Process in the Reproductive Organs of C57Bl/6 Female Mice with Experimental Tuberculosis.

Intravenous infection of C57Bl/6 female mice with M. tuberculosis H37Rv led to involvement of the lungs and dissemination of the tuberculous infection to the abdominal and pelvic organs. M. tuberculosis were detected in the lungs and spleen in 14, 35, and 90 days and in the uterine horns in 90 days after infection. Morphological analysis of organs showed successive development of exudative necrotic tuberculosis of the lungs, acute and chronic nonspecific inflammation in the reproductive organs (vagina, uterus, and uterine horns). The inflammatory process in the reproductive organs was associated with the development of anaerobic dysbiosis, that was most pronounced in 35 days after infection. Antituberculous therapy was followed by reduction of M. tuberculosis count in the lungs and spleen in 60 and 90 days after infection, eliminatation of M. tuberculosis in the uterine horns, arrest of nonspecific inflammation in female reproductive organs, recovery of the balance between aerobic and anaerobic microflora, and development of candidiasis of the urogenital mucosa.

Antibacterial Effects of Liposomes Containing Phospholipid Cardiolipin and Fluoroquinolone Levofloxacin on Mycobacterium tuberculosis with Extensive Drug Resistance.

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.

The Role of Neutrophil Granulocyte Ultrastructures in the Formation of Extracellular Traps.

We demonstrated the contribution of cytoskeleton, calcium channels, energy metabolism, and membrane of neutrophils in the formation of neutrophil extracellular traps.

Draft Genome Sequences of Two Pyrazinamide-Resistant Clinical Isolates, Mycobacterium tuberculosis 13-4152 and 13-2459.

We report draft genome sequences of two pyrazinamide (PZA)-resistant isolates, Mycobacterium tuberculosis 13-4152 and 13-2459. Isolate 13-4152 is PZA resistant, though it lacks mutations in known genes of PZA resistance. The comparative analysis of these genomes with those stored in GenBank revealed unique mutations, which may elucidate new mechanisms of PZA resistance.

[Synthesis and biological properties of α-thymidine 5'-aryl phosphonates].

The interaction of CDI-activated diethyl phosphonoacetate with methyl 4-aminobenzoat or 3,5-difluoromethylphenylamine followed by treatment with Me3SiBr in DMF led to N-aryl aminocarbonylmethyl phosphonates and their ethyl esters. Their coupling with 3'-acetyl-α-thymidine followed by removal of the acetyl groups gave (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl-, aminocarbonyl- and carboxy)phenyl]-aminocarbonylmethyl phosphonates, (α-D-thymidine-5'-il)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. The phosphonates were stable in different conditions, low cytotoxic (in Vero and K562 cells) and were able to penetrate into K562 cells. The only ethyl ester of (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl)phenyl]-aminocarbonylmethyl phosphonate in high concentration (200 µg/mL) inhibited in vitro the growth of laboratory sensitive strain of Mycobacterium tuberculosis H37Rv.

Performance of Cepheid ® Xpert MTB/RIF ® and TB-Biochip ® MDR in two regions of Russia with a high prevalence of drug-resistant tuberculosis.

The purpose of this study was to assess the performance of Cepheid® Xpert MTB/RIF® ("Xpert") and TB-Biochip® MDR ("TB-Biochip"). Sputum specimens from adults with presumptive tuberculosis (TB) were homogenized and split for: (1) direct Xpert and microscopy, and (2) concentration for Xpert, microscopy, culture [Lowenstein-Jensen (LJ) solid media and Mycobacteria Growth Indicator Tube® (MGIT)], indirect drug susceptibility testing (DST) using the absolute concentration method and MGIT, and TB-Biochip. In total, 109 of 238 (45.8 %) specimens were culture-positive for Mycobacterium tuberculosis complex (MTBC), and, of these, 67 isolates were rifampicin resistant (RIF-R) by phenotypic DST and 64/67 (95.5 %) were isoniazid resistant (INH-R). Compared to culture of the same specimen, a single direct Xpert was more sensitive for detecting MTBC [95.3 %, 95 % confidence interval (CI), 90.0-98.3 %] than direct (59.6 %, 95 % CI, 50.2-68.5 %) or concentrated smear (85.3 %, 95 % CI, 77.7-91.1 %) or LJ culture (80.8 %, 95 % CI, 72.4-87.5 %); the specificity was 86.0 % (95 % CI, 78.9-91.3 %). Compared with MGIT DST, Xpert correctly identified 98.2 % (95 % CI, 91.5-99.9 %) of RIF-R and 95.5 % (95 % CI, 85.8-99.2 %) of RIF-susceptible (RIF-S) specimens. In a subset of 104 specimens, the sensitivity of TB-Biochip for MTBC detection compared to culture was 97.3 % (95 % CI, 91.0-99.5 %); the specificity was 78.1 % (95 % CI, 61.5-89.9 %). TB-Biochip correctly identified 100 % (95 % CI, 94.2-100 %) of RIF-R, 94.7 % (95 % CI, 76.7-99.7 %) of RIF-S, 98.2 % (95 % CI, 91.4-99.9 %) of INH-R, and 78.6 % (95 % CI, 52.1-94.2 %) of INH-S specimens compared to MGIT DST. Xpert and Biochip were similar in accuracy for detecting MTBC and RIF resistance compared to conventional culture methods.

Carbocyclic analogues of inosine-5'-monophosphate: synthesis and biological activity.

9-(4'-Phosphonomethoxy-2'-cyclopenten-1'-yl)hypoxanthine and 9-(4'-phosphonomethoxy-2',3'-dihydroxycyclopenten-1'-yl)hypoxanthine were synthesized as isosteric carbocyclic analogues of inosine-5'-monophosphate. The synthesized compounds were shown to be capable of inhibiting the activity of human type II inosine-5'-monophosphate dehydrogenase (IMPDH II) (IC(50 )= 500 µM) and to have no significant effects on the growth ofMycobacterium tuberculosis.

Molecular typing of Mycobacterium tuberculosis circulated in Moscow, Russian Federation.

The present study investigates epidemiological diversity and multidrug resistance spreading among Mycobacterium tuberculosis strains circulating in Moscow, Russian Federation. Among 115 M. tuberculosis strains selected randomly from the sputum of epidemiologically unrelated tuberculosis (TB) patients, multidrug-resistant (MDR) strains predominated. Mutations in the RRDR of the rpoB gene were detected in 64 (83.1%) of 77 rifampicin (RIF)-resistant strains. The Ser531→Leu substitution was prevalent among them (76.5%). Aberrations in the Ser315 codon of katG and/or in the inhA promoter region were found in 79 (84.0%) of 94 isoniazid (INH)-resistant strains. Strains belonging to the Beijing family prevailed. Seventy-one different patterns were identified using the 24-VNTR loci typing scheme. Three main 24-loci VNTR clusters included 34 strains which belonged to the Beijing family. The spoligotyping and 24-loci VNTR typing combination demonstrated maximal discriminatory power. Among the Beijing strains, the MDR phenotype was revealed more frequently than among the others. High genetic heterogeneity of the studied population was shown by the assessment of VNTR loci variability in the analyzed group and in the strains from other parts of Russia. Comparison of the 24-VNTR locus typing and spoligotyping data with revealed resistance-associated mutation allows us to make a suggestion that the active transmission of MDR strains and the independent appearance of drug resistance during chemotherapy occurred in the studied population simultaneously.

[Regulatory effect of complex of natural cytokines and antimicrobial peptides on murine macrophages infected with multidrug-resistant Mycobacterium tuberculosis].

AIM: To study the effect of complex of natural cytokines and antimicrobial peptides (CNCAP) included in preparation Superlymph on growth of multidrug-resistant Mycobacterium tuberculosis CN-37 on the modem of murine peritoneal macrophages (MPh) cultivated in vitro. MATERIALS AND METHODS: Effect of CNCAP on peritoneal MPh of tuberculosis-susceptible mice C57BL/6 infected by M. tuberculosis CN-37 was studied using ex vivo model. Macrophages were preliminary incubated with CNCAP during one day. M. tuberculosis growth was assessed on 7th day by PCR. RESULTS: Preliminary incubation of infected MPh with CNCAP resulted in inhibition of M. tuberculosis CN-37 growth. CONCLUSION: Superlymph activates macrophages which lead to enhanced bactericidal action of MPh on M. tuberculosis CN-37.

New 5-modified pyrimidine nucleoside inhibitors of mycobacterial growth.

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 2'-deoxy-, 3'-azido-2',3'-dideoxy-, and 3'-amino-2',3'-dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M. tuberculosis in vitro. 5-Dodecyloxymethyl-2'-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 level of cytokines in the ex vivo infection of murine macrophages with Mycobacterium tuberculosis complex].

To study whether the genotype of a mycobacterial strain might affect the ability of macrophages (MP) to produce the key cytokines, we determined the synthesis of interferon-gamma (IFN-gamma), tumor necrosis factor-gamma (TNF-gamma), and interleukin-6 (IL-6) in the infection of murine MP with M. tuberculosis H37Rv and M. bovis BCG, the strains that belong to the same genetic group (M. tuberculosis complex) but are opposite in virulent properties. MP infection with a virulent and attenuated M. tuberculosis complex strain was shown to differently affect the synthesis of IFN-gamma, TNFaalpha-, and IL-6. MP infection with M. tuberculosis H37Rv activated the generation of IFN-gamma and that with M. bovis BCG substantially increased the levels of TFN-alpha and IL-6. The findings suggest that this model may be used to investigate the specific features of mycobacterial strains of various genotypic clusters with eukaryotic cells.

[Detection of mutations in codon 306 of the embB gene for molecular genetic characterization of clinical Mycobacterium tuberculosis strains].

A total of 254 Mycobacterium tuberculosis strains were used in the study. Among them, there were 183 ethambutol (EMB)-resistant strains, 13 multidrug resistant ones, but EMB-sensitive, and 39 strains sensitive to rifampicin (RIF), isoniazid (INZ), and EMB. All the strains were analyzed for genetic changes in three loci: embB306, rpoB, and katG/inhA promoter, which were associated with the formation of resistance to EMB, RIF, and INZ, respectively. The Mycobacterium tuberculosis strains were obtained from pulmonary tuberculosis patients living in the Central Region of the Russian Federation. Resistance to RIF, INZ, and EMB was revealed by the absolute concentration test. The inhibitory concentration (IC) of EMB was determined for all the strains. Genetic changes in the above loci were estimated by mini-sequencing, followed by mass-spectrometry recording MALDI-TOF products. The relative low frequency of embB306 mutations was observed among the EMB-resistant strains (about 41.5%). Mutations in codon 306 were detected only in strains with EMB IC > or = 2 mg/L. A statistical significant association was found between the frequency of embB306 mutations and the multidrug resistant phenotype. A combination of these mutations with the traditional genetic markers of multidrug resistance may be used for the more effective detection of multidrug-resistant strains.

Effects of liposomes of different lipid composition on in vitro growth of Mycobacterium tuberculosis H37Rv.

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.

[Biological properties of M. Tuberculosis W cluster strains].

To study the biological characteristics of M. tuberculosis W cluster strains, the authors carried out in vitro, ex vivo, and in vivo experiments on 18 clinical strains and 2 laboratory ones, which had been clustered by a standardized methodology. Comparison was made in experiments in vitro and in macrophageal inoculation (ex vivo) from the 5,6-[3H]-uracil incorporation that reflected mycobacterial replication. The in vivo experiments estimated mycobacterial survival and cultivation and the lung pathomorphological pattern of infected animals. The results showed that M. tuberculosis W cluster strains had in vitro the mean multiplication rate, showing a high viability in the macrophages; the in vivo experiments demonstrated that the W cluster strains did not differ in virulence and might be both more and less virulent than the laboratory strain H37Rv. Notwithstanding the fact that the assumption on the hypervirulence of M. tuberculosis W cluster has not been supported, the distinguishing characteristic of the strains of this cluster is their enhanced capacity to survive in the macrophages no matter what infective dose.

Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips.

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.

[Inhibiting effect of complex of natural cytokines and cationic antimicrobial peptides on the growth of Mycobacterium tuberculosis H37Rv in vitro].

AIM: To assess direct antimicrobial effect of complex of natural cytokines (CNC) and antimicrobial peptides (Syperlymph preparation; CNC) on Mycobacterium tuberculosis H37Rv (H37Rv) and effect mediated by macrophages (MP) treated with the preparation. MATERIALS AND METHODS: Direct effect of CNC was studied during cultivation of H37Rv in the presence of preparation, whereas indirect effects--during simultaneous cultivation of H37Rv and mice peritoneal MP C57B1/6. Assessment of growth was performed on the 7th day using PCR. RESULTS: It was shown that CNC directly inhibits growth of H37Rv in vitro. Cultivation of H37Rv in culture of MP resulted in inhibition of M. tuberculosis. The most evident inhibition was noted after extension of time of preliminary treatment of MP with Syperlymph and simultaneous increase of its concentration. CONCLUSION: Antimycobacterial effect of Syperlymph preparation related to complex effect of cytokines and antimicrobial peptides directed to M. tuberculosis and macrophages, which forms the conditions for killing of mycobacteria.

[Impact of M. tuberculosis genotype on survival in mice with experimental tuberculosis].

To study the virulence of Mycobacterium tuberculosis strains of the W cluster, C57B1/6 mice were intravenously inoculated with a lethal dose (5x10(6) CFU) of 14 clinical M. tuberculosis strains (11 drug-sensitive and 3 multidrug-resistant) belonging to different RFDP IS6110 genotypic clusters and two laboratory M. tuberculosis strains H37Rv and H37Ra. The virulence was evaluated by the survival of mice after infection and by the trends in body weight loss. The study indicated that the mice inoculated with different M. tuberculosis strains differed in survival rates and in the trend in body weight loss. A minor HD cluster strain and 2 non-clustered strains were most virulent, next were 2 AI cluster strains. W cluster strains had both higher (n = 2) and lower (n = 3), and comparable (n = 2) H37Rv virulence. A KQ cluster strain had the least virulence. An attenuated H37Ra strain caused no animal death. Inoculation with three multidrug-resistant strains belonging to the W cluster (n = 2) and one non-clustered strain demonstrated no relationship of virulence to the sensitivity of a strain to antituberculous agents. The findings argue against the opinion on W cluster M. tuberculosis strains as hypervirulent.