This study presents an analysis of M. tuberculosis growth data obtained using the BACTEC MGIT 960 system and respective mathematical models. The system is based on the detection of a decrease in oxygen level in the broth due to the bacterial respiration. It is shown that recordings sampled with a 1 hour rate provide an opportunity to distinguish between the oxygen consumption of growing cells and active cells division when the density of micro-organisms is sufficient to enter into the synchronized division mode. More specifically, the growth of culture is continuous only with large initial dilutions; otherwise, there are jumps between different growth stages with a time interval of 13–15 h. The combination of the oxygen-quenching kinetics for an analytic reagent and the population growth kinetics resulted in a mathematical model, which consists of mixing Verhulst's and Gompertz's models. The parameters of such mixing and switching between the models' prevalences are discussed with respect to oxygen uptake reactions reflected in the changes in the experimentally registered fluorescence level.

The Mycobacterium tuberculosis (MbT) drug resistance remains a serious problem to global public health and usually leads to a lethal outcome in patients with such strain MbT. Precise and rapid identification of a specific MbT strain is vital for the appointment of anti-tuberculosis therapy. We continue investigation( ) of deactivated MbT strains of the Beijing family by Raman spectroscopy method and focus on glutathione (GSH) as a potential biomarker for strains identification. Strains under the study have different drug sensitivity: sensitive, multi, and extensively drug resistant. Samples were investigated by Raman spectrometry with He-Ne (633 nm) laser excitation source in the "fingerprint" region. As a result, GSH vibrational bands for potential strain discrimination were determined. These differences can be described for drug-sensitive and drug-resistant strains taken of pulmonary and extra-pulmonary forms of tuberculosis. We suppose that the obtained results can be useful for new diagnostic tools development for identification of different MbT strains in clinical medicine and research practice.

Over the past decade Raman spectroscopy has been successfully used to study the cell structure of various pathogens, including M. tuberculosis (MbT). It is especially important due to the high ability of pathogen to become resistant to anti-tuberculosis drugs usually leading to a lethal outcome. In the present study we investigate deactivated MbT strains with different drug sensitivity (extra-, multi- and sensitive drug resistance) of the Beijing family. Samples have been obtained from patients suffering from pulmonary and extra-pulmonary forms of tuberculosis. Samples have been investigated by Raman spectrometry using a diode-pumped solid-state argon (514 nm) laser excitation source in the high-wavenumber region. As a result, protein and lipid vibrational bands for strain discrimination with various drug resistances have been determined. The obtained results can be used as fast diagnostic tool development for screening MbT strains in clinical medical practice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Starting from the structure of antimycobacterial screening hit OTB-021 which was devoid of activity against ESKAPE pathogens, we designed, synthesized and tested two mutually isomeric series of novel simplified analogs, 2- and 4-(3-nitro-1,2,4-triazol-1-yl)pyrimidines, bearing various amino side chains. These compounds demonstrated a reverse bioactivity profile being inactive against M. tuberculosis while inhibiting the growth of all ESKAPE pathogens (with variable potency patterns) except for Gram-negative P. aeruginosa. Reduction potentials (E-1/2, V) measured for selected compounds by cyclic voltammetry were tightly grouped in the -1.3--1.1 V range for a reversible single-electron reduction. No apparent correlation between the E-1/2 values and the ESKAPE minimum inhibitory concentrations was established, suggesting possible significance of other factors, besides the compounds' reduction potential, which determine the observed antibacterial activity. Generally, more negative E-1/2 values were displayed by 2-(3-nitro-1,2,4-triazol-1-yl)pyrimidines, which is in line with the frequently observed activity loss on moving the 3-nitro-1,2,4-triazol-1-yl moiety from position 4 to position 2 of the pyrimidine nucleus.

The rezazurin assay for screening of bacterial growth remains the most popular method for express diagnostic tests. It is based on the change of colour and uorescence properties of the respective dye due to bacterial breathing but its conventional quantitative practical implementation requires a relative expensive and cumbersome spectrophotometric equipment. As an alternative approach, we propose to shift a focus from hardware to software. Preliminary results announced in this work show that the processing RGB colour channels for simple photographic images of resazurin assay plates with mycobacterium tuberculosis provides an opportunity to reconstruct the target growth curve with an accuracy comparable with results obtained using the conventional photometric uorescence curves.

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.

Nowadays proteomics is one of the major instruments for editing and correcting annotation of genomic information. The correct genome annotation is necessary for omics studies of clinically relevant pathogens like Mycobacterium tuberculosis as well as for the progress in drug design and in silico biology. Here, we focused on the proteogenomic analysis of W-148 strain belonging to the Beijing B0/W148 cluster. This cluster, also known as a "successful" clone possesses unique pathogenic properties and has a unique genome organization. Taking into account high similarity of cluster strains at the genomic level we analyzed MS/MS dataset obtained for 63 clinical isolates of Beijing B0/W148. Based on H37Rv and W-148 annotations we identified 2546 proteins representing more than 60% of total proteome. A set of peptides (n = 404) specific for W-148 was found when compared with H37Rv. Start sites for 32 genes were corrected based on the combination of LC-MS/MS proteomic data with genomic six-frame translation. Additionally, we have shown the presence of peptides related to 10 genes earlier known as "pseudogenes". Significance: Mycobacterium tuberculosis is one of the most dangerous pathogens. Phylogenetically, it may be divided into major lineages and among them, lineage 2 (predominantly Beijing genotype) one of the most successful lineages with an increasing prevalence in the global population. At the same time, strains of the Beijing B0/W148 cluster, a "successful" clone of Mycobacterium tuberculosis possess even more interesting features. Only one complete genome of this cluster, W-148, present in the NCBI database (CP012090.1) and it demonstrates a number of significant differences from the well-known reference genome H37Rv. For the W-148 strain many genes are annotated as "pseudo" and no attempts were made to correct this. Thereby, in this study, we have conducted a proteomic analysis of the cluster strains and corrected current genome annotation. We hope that the data obtained will help to increase the quality of identifications in proteomic and transcriptomic analysis of M. tuberculosis Beijing B0/W148 cluster strain in subsequent studies.

Mycobacterium tuberculosis Beijing genotype strains comprise 5080% 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.

Mycobacterium tuberculosis Beijing B0/W148 is one of the most widely distributed clusters in the Russian Federation and in some countries of the former Soviet Union. Recent studies have improved our understanding of the reasons for the "success" of the cluster but this area remains incompletely studied. Here, we focused on the system omics analysis of the RUS_B0 strain belonging to the Beijing B0/W148 cluster. Completed genome sequence of RUS_B0 (CP020093.1) and a collection of WGS for 394 cluster strains were used to describe the main genetic features of the population. In turn, proteome and transcriptome studies allowed to confirm the genomic data and to identify a number of finds that have not previously been described. Our results demonstrated that expression of the whiB6 which contains cluster-specific polymorphism (a151c) increased almost 40 times in RUS_B0. Additionally, the level of ethA transcripts in RUS_B0 was increased by more than 7 times compared to the H37Rv. Start sites for 10 genes were corrected based on the combination of proteomic and transcriptomic data. Additionally, based on the omics approach, we identified 5 new genes. In summary, our analysis allowed us to summarize the available results and also to obtain fundamentally new data.

A selectively antimycobacterial compound belonging to the nitrofuran class of antimicrobials has been developed via conjugation of the nitrofuran moiety to a series of spirocyclic piperidines through an amide linkage. It proved to have comparable activity against drug-sensitive (H37Rv) strain as well as multidrug-resistant, patient-derived strains of Mycobacterium tuberculosis. The compound is druglike, showed no appreciable cytotoxicity toward human retinal pigment epithelial cell line ARPE-19 in concentrations up to 100 μM and displayed low toxicity when evaluated in mice.

Tuberculosis is one of the most widespread worldwide diseases heavily affecting society. Among popular modern laboratory tests for mycobacterial growth, the resazurin assay has certain advantages due to its effectiveness and relatively low cost. However, the high heterogeneity of the mycobacterial population affects the average growth rate. This fact must be taken into account in a quantitative interpretation of these tests’ output—fluorescence growth curves—related to the population growth of viable mycobacteria. Here, we report the spectrophotometric data obtained via the resazurin assay for the standard reference strain of Mycobacterium tuberculosis H37Rv for different initial dilutions and generation numbers of the culture, as well as their primary processing from the point of view of the stochastic multiplicative growth model. The obtained data, which indicate an accelerated (instead of linear) growth of the population density logarithm between the end of the lag phase and the saturation, provide evidence of the importance of the growth rates’ stochasticity. An analysis of the curve fits resulted in an estimation of the first two moments of the growth rates’ probability distributions, showing its relevance to vital processes for mycobacterial culture.

The drug resistance of Mycobacterium tuberculosis (MbT) remains a serious challenge to global public health as extensively drug resistant tuberculosis usually leads to a lethal outcome. In the case of diseases with resistant strains, it is extremely important to identify a specific strain as soon as possible in order to start an adequate therapy. One such diagnostic method could be the Raman spectroscopy method. We investigated M. tuberculosis strains of the Beijing family deactivated by heating, with different drug sensitivity: sensitive, multi-and extensively drug resistant. Samples, obtained from patient specimens were investigated by Raman spectrometry with the He-Ne (632.8 nm) laser excitation source. As a result, a set of optimal experimental parameters for strain discrimination were determined based on the cell-wall spectral differences. These differences distinguish between drug-sensitive and drug-resistant strains as well as between strains isolated from material samples taken from patients suffering from pulmonary and extra-pulmonary forms of tuberculosis. We suggest that the obtained results allow the usage of the Raman spectroscopy as a fast diagnostic tool for determining various strains in clinical medical practice.

This data article contains Raman experimental data, obtained with Horiba Jobin-Yvon LabRam HR800 spectrometer (Japan), which can be used for rapid identification of Mycobacterium tuberculosis (MbT) bacteria (Beijing clade) in vitro. Data present analyzed Raman spectra of bacterial cells with various drug resistances obtained from pulmonary and extra pulmonary samples. Data can provide information about characteristic maxima of different structures in biological cell.

Within the general nitrofuran carboxamide chemotype, chimera derivatives incorporating diversely substituted imidazoles attached via an alkylamino linker were synthesized. Antimycobacterial evaluation against drug-sensitive M. tuberculosis H37Rv strain identified five active druglike compounds which were further profiled against patient-derived M. tuberculosis strains in vitro. One of the compounds displayed promising potent activity (MIC 0.8 μg/mL) against one of such strains otherwise resistant to such first- and second-line TB therapies as streptomycin, isoniazid, rifampicin, ethambutol, kanamycin, ethionamide, capreomycin and amikacin. The compound was shown to possess low toxicity for mice (LD50 = 900.0 ± 83.96 mg/kg) and to be similarly efficacious to etambutol, in the mouse model of drug-sensitive tuberculosis, and to neurotoxic cycloserine in mice infected with MDR tuberculosis.

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.

Comparative proteomic profiling of M. tuberculosis H37Rv strains cultured on two different nutrient media, Lowenstein-Jensen and Middlebrook 7H11, was performed using a label-free LC-MS/MS approach. It was shown that results obtained from two media possessed high convergence. The only difference was observed in the representation of fumarate reductase FrdB, its abundance was higher in the mycobacterial cells cultured on the Lowenstein-Jensen medium. The correlation analysis of biological repeats revealed the high convergence of the results obtained using the Middlebrook 7H11 medium. Thus, we can conclude that the use of the Middlebrook 7H11 medium is most appropriate in the scientific laboratory.

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.