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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.

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.

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.

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.

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.