Genome-Wide Mycobacterium tuberculosis Variation (GMTV) Database: A New Tool for Integrating Sequence Variations and Epidemiology

Background Tuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome. Description Here we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes. Conclusions Implementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains

MYCOBACTERIUM TUBERCULOSIS VIRULENCE

This review presents recent data on the genetic determinants and the virulence factors of Mycobacterium tuberculosis (MBT). The mechanisms of adaptation of the pathogen in the host organism and alterative manifestations are described as well as the relationship of virulence, drug resistance and the genetic affiliation of MBT are analyzed in thearticle. It was demonstrated the clinical and prognostic significance of Mycobacterium tuberculosis virulence. The cyclic changes in MBT virulence coincide with the rise and fall of the incidence of tuberculosis has been established. Some virulence factors are targets to create fundamentally new anti-TB drugs

THE GROWTH RATE PHENOTYPIC PROPERTY OF MYCOBACTERIUM TUBERCULOSIS CLINICAL STRAINS: DEPENDENCE ON TUBERCULOSIS LOCALIZATION, TREATMENT, DRUG SUSCEPTIBILITY

The phenotypic properties of the M. tuberculosis strains obtained from patients with pulmonary or extra-pulmonary tuberculosis are  determined by a complex set of factors: the genetic characteristics  of the pathogen, its ability to adapt in vivo and in vitro, the influence of the host’s immune system and chemotherapy. The growth rate as  the phenotypic property is the most accessible for the study of the  host-pathogen relationships at the level of host/strain population  interactions. The aim of the study is to assess in vitro of the growth  rate of M. tuberculosis strains isolated from patients with pulmonary  and extra-pulmonary tuberculosis: untreated and treated (with  surgical and non-surgical treatment) and also sensitive and resistant isolates in comparison with the reference strain H37Rv. To estimate  the growth rate of 116 clinical isolates we have used the modified  method originally developed by von Groll and co-authors: to get the  bacteria growth curve the fluorescence intensity of growing strains  (with indicator resazurin) has been measured daily for 8 days in 96- well plate. The growth rate is determined as the slope of the growth  curve. The mean values of the growth rate have been calculated in  the following groups of patients: 1 — untreated patients with  pulmonary tuberculosis (PT), respiratory material; 2 — non-surgical  treated PT patients, respiratory material; 3 — surgical treated PT  patients (mainly with chronic and hyperchronic process), respiratory  material; 4 — patients like in 3rd group, surgical material; 5 — bone  and joint tuberculosis (BJT), surgical material. In addition, groups of  sensitive and resistant strains have been examined, but there are no  significant differences in growth rates. It has been obtained that  the growth rate of strains isolated from the PT patients is higher than in BJT patients: it can be explained less favorable  conditions for the pathogen vegetation in the BJT. In the case of a  closed tuberculous lesion where the pathogen transmission to  another host is impossible, then the selection of strains with the  property to survive in the tissues of the osteoarticular system is  impossible too, therefor it should be observed only an adaptation of  the pathogen strain population to the individual host. The growth  rate of isolates from untreated PT patients is higher than that of the  treated ones. Comparison of the growth parameters of only MDR  strains 1–5 groups to eliminate the influence of the  sensitivity/resistance has resulted in the same conclusions. We  suggest that the decrease in the growth rate of strains from the  treated PT patients is in not only result of the treatment, but also is  conditioned by adaptation of the pathogen to its external  environment, which is the internal environment of the  macroorganism. To confirm this assumption, the bacterial load of  1,083 diagnostic specimens grouped in a similar manner has been  estimated, taking into account only MDR/XDR strains. In the group  of treated patients the frequency of high bacterial load (CFU ≥ 100)  reached 52.5–63.8% that shows the conserved fitness of bacteria in  such patients. The mean values of the growth rate of the strain  H37Rv non-adapted to the macroorganism (due to numerous  passages on artificial media) are higher than in all groups of clinical  strains. Thus, heterogeneity of phenotypic properties of M.  tuberculosis clinical strains on the basis of growth rate has been  obtained. The growth rate of M. tuberculosis clinical strains is  depended on the tuberculosis localization (PT, BJT) and on the joint  effect of patient treatment and pathogen adaptation to the host

DRUG RESISTANCE, VIABILITY AND VIRULENCE IN VITRO ОF MYCOBACTERIUM TUBERCULOSIS STRAINS OF DIFFERENT GENOTYPES

Abstract. The drug resistance, mutation spectrum caused resistance to rifampicin and isoniazid, viability, cytotoxicity were studied as well as 111 clinical isolates of Mycobacterium tuberculosis (MBT) were genotyped. Spoligotyping revealed 28 spoligotypes; the greatest number of strains belonged to genetic families Beijing and LAM. The typing of 59 strains of spoligotype SIT1 (Beijing) allowed to differentiate 19 variants of IS6110-RFLP-profiles: 13 of them were individual and 6 were presented by clusters. The clusters А0 and В0 included the greatest number of M. tuberculosis strains — 21 (35,6%) and 17 (28,8%) accordingly. The high frequency of MLU and SLU of Beijing strains was associated with mutations rpoB Ser531→Leu and katG Ser315→Thr. The strains of M. tuberculosis belonged to another genetic families (T, H, Ural, U) have shown drug resistance more often. The level of resistance to isoniazid in vitro in MLU/SLU strains of MBT of different genotypes, especially, was high in case of mixed mutations such as katG Ser315→Thr и inhA_T15. The rates of viability and cytotoxicity of MBT strains of studied genotypes with different spectrum of mutations and phenotypic drug resistance were not substantially distinguished

MYCOBACTERIUM TUBERCULOSIS BIOLOGICAL PROPERTIES AND CHARACTERISTICS OF THE INFLAMMATORY REACTION IN PATIENTS WITH INFILTRATIVE PULMONARY TUBERCULOSIS

Host-pathogen relations were analyzed on basis of the results of examinations of 42 patients with newly diagnosed, previously untreated infiltrative pulmonary tuberculosis (IPT) with regard for the biological characteristics of Mycobacterium tuberculosis (MBT) and the biomarkers of the inflammatory reaction — the acute phase proteins (APPs). The genotypes of MBT (Beijing and the others) and the efficacy of threeonth antituberculosis therapy were used as the grouping factors. Genotype Beijing MBT patients were significantly often characterised by multiple drug resistant, had widespread pulmonary damage and association of MBT cytoxicity with the effect of therapy. The patients with the best postreatment effect had initial (pretreatment) APPs levels in the range of the referent ones. These conclusions were confirmed by analysis of the correlation pleads of the characteristics of the “host-pathogen” system and their discriminant analysis with regard for MBT genotypes and results of antituberculosis three-month therapy. It was shown that the constellation of four host’s APP characteristics (haptoglobin, ceruloplasmin, elastase and adenoaminase activities) has above 90% prognostic efficacy of treatment in spite of MBT genotype. It is suggested that in IPT patients the first component of the “host-pathogen” system reflecting the reaction of the patient’s organism to the MBT induced inflammatory process is more prognostically important

DRUG RESISTANCE OF MYCOBACTERIUM TUBERCULOSIS IN DIFFERENT LOCALIZATIONS OF THE DISEASE

The long-term Mycobacterium tuberculosis drug resistance monitoring data were obtained in St. Petersburg Research Institute Phthisiopulmonology for four periods of observation (I — 1984–1988 years, start period; II — 1996–2000 years, the period of acute epidemic situation and the sharp rise in drug resistance; III — 2007–2011 years, the period of relative stabilization; IV — 2012–2014 years, the final period). Totally 2267 strains from patients with pulmonary tuberculosis, treated previously and with chronic process, and 691 strains from patients with extrapulmonary tuberculosis were studied. Researches of drug resistance were made by indirect method of absolute concentrations in the medium Lowenstein–Jensen and in the automated system ВАСТЕС™ MGIT™ 960 (Becton Dickinson, USA). It shows a steady increase of the overall drug resistance, which has reached in the last three years 90.1% in pulmonary tuberculosis. At the same time there was a sharp worsening of the structure of drug resistance due to the growth of multidrug (MDR) and extensively drug-resistant (XDR), which by 2014 accounted for 81.9 vs 28.5% in 1984–1988. In extrapulmonary tuberculosis the growth of drug resistance of Mycobacterium tuberculosis continues to outpace: overall drug resistance increased from 39.4% (1984–1988) to 80.2% (2012–2014), reaching figures that are comparable to those of pulmonary tuberculosis. At the same time there was an even more rapid change for the worse of its structure due to the increase of MDR/XDR strains: from 10.5% in the first period to 69.5% in the fourth. This is due to the more frequency of existing in locus of extrapulmonary tuberculosis of highly adaptive multiresistant strains of genotype Beijing: in tuberculous spondylitis, which is the most severe and frequent form of osteoarticular tuberculosis, 70 of the 78 (89.7%) Mycobacterium tuberculosis isolates with MDR/XDR belong to this genotype. In pulmonary tuberculosis the XDR growth rate exceed those in extrapulmonary tuberculosis: from 26.8% (III period) to 39.5% (IV period) and, accordingly, from 8.0 to 8.6%. The situation with drug resistance in all localizations of the disease may be characterized as extremely tense, which can lead to unpredictable consequences, if promptly one not take appropriate measures

In vivo virulence of Beijing genotype Mycobacterium tuberculosis

Mycobacterium tuberculosis Beijing genotype strains comprise 50–80% in Russian Federation, which are divided into the main B0/W148, CladeA, and CAO clusters based on VNTR and SNP analysis. It should be noted that such phylogenetically highly close MTB strains belong to the modern Beijing family, generally demonstrating high transmissibility, association with drug resistance, and prevalence among patients with severe forms of the disease. However, studies on MTB genetic cluster strain-related virulence are scarce and contradictory. Here, we investigated virulence of diverse Mycobacterium tuberculosis strains belonging to the B0/W148, CladeA and CAO clusters and nonclustered strain NK of the Beijing family as well as laboratory strain H37Rv in C57BL/6 mice. It was found that mice infected with NK and B0/W148 vs. CladeA strains revealed the peak and the lowest mortality, respectively, while assessing survival rate in various groups (20 mice per MTB strain examined). Analyzing experimental data in mice demonstrated that all MTB strains were able to cause typical tuberculosis-related pathogenic signs. In particular, time-dependent evaluation of pathological changes (on 1, 3, 7, 14, 21, 28, 60 and 120 day post infection) in the lungs and spleen revealed significant differences among various strains. Tuberculosis progression was observed in the mice infected with B0/W148 and NK strains, whereas CladeA, CAO and H37Rv strains resulted in stabilized course and less marked organs damage. Moreover, we found that bacterial load after infection with Beijing family clustering strains was lower compared to that of the reference H37Rv strain, except NK strain demonstrating the peak bacterial load among the Beijing family comparable to H37Rv strain at 120 dpi. Thus, it was found that the level of virulence between most virulent B0/W148 cluster strain vs. NK strain was similar. Overall, the data obtained indicate that Beijing genotype strains are characterized by a diverse range of phenotypic virulence in vivo

Synthesis and antimicrobial activity of adamantyl substituted pyridoxine derivatives

© 2019 Bentham Science Publishers Background: Adamantane derivatives possess multiple pharmacological activities such as antiviral, anticancer, antimycobacterial, antidiabetic, antiparkinsonian and others. The interest of medicinal chemists in adamantane compounds is due to their unique spatial structure, high lipophilicity, and carbon cage rigidity. As a result, these molecules can easily penetrate biological lipid membranes and often have unique target-specific activity profile. Another pharmacophore studied in this work is pyridoxine (vitamin B6). Pyridoxine plays highly important roles in living cells as a key cofactor of many enzymes. On the other hand, its molecular scaffold is a valuable structural platform which has led to the development of several launched drugs (Pyritinol, Pirisudanol, Cycletanine, Mangafodipir) and a wide number of preclinical and clinical drug candidates. Objective: The objective of this study is a synthesis of pyridoxine-adamantane and pyridoxinecyclooctane dipharmacophore molecules. The underlying idea was to assess the antibacterial and antiviral potential of such dipharmacophores, based on multiple examples of promising antiinfective agents which have in their structures adamantane and pyridoxine moieties. Another specific reason was to explore the ability of pyridoxine pharmacophore to suppress the potential of microbial pathogens to develop resistance to drug molecules. Methods: In this study, a series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkyl amines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. All synthesized compounds have been tested for their in vitro activity against M. tuberculosis H37Rv strain and H3N2 (A/Aichi/2/68) influenza virus. Results: Series of pyridoxine-adamantane and pyridoxine-cyclooctane dipharmacophore molecules were synthesized based on reactions of three different cycloalkylamines with the corresponding electrophilic derivatives of pyridoxine aldehydes, chlorides and acetates. Reaction of cycloalkylamines with pyridoxine derivatives, in which meta-hydroxyl and ortho-hydroxymethyl groups are protected by acetyl groups, represents a useful alternative to reductive amination of aldehydes and nucleophilic substitution of alkyl halides. According to a tentative mechanism, it proceeds via paraand ortho-pyridinone methides which readily react with nucleophiles. None of the synthesized dipharmacophore compounds showed activity against M. tuberculosis H37Rv strain. At the same time, three compounds demonstrated some antiviral activity against H3N2 (A/Aichi/2/68) influenza virus (EC50 52-88 μg/mL) that was comparable to the activity of Amantadine, though lower than the activity of Rimantadine. The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane. Conclusion: The results of this work can be useful in the design of physiologically active derivatives of pyridoxine and adamantane