Vol 95, No 2 (2018)

Aim. The work was performed with the purpose to study an inhibitory action of millimolar concentrations of divalent metal ions, which differ by primary mechanisms of their toxicity, on the culture of S.pyogenes. Materials and methods. Suspensions of S.pyogenes bacteria which contained 10⁸ CFU/ml were sown by the lawns into the standard Petri dishes coated with the supplemented Nutrient Agar. 30 min later the salt solutions of divalent metals were added by the 5 pl drops on the surfaces of the lawns with use of 36-channel stamp replicator. The salt solutions contained the metals tested at the concentrations ranged between 5x10^-3 M to 5x10^-1 M. Then the dishes with bacterial cultures were incubated for 24 hrs at 37°C followed by measuring diameter of the area of culture growth inhibition. The probes of material obtained from the centers of the stunting areas were passed into the centrifuge tubes with the supplemented Nutrient Broth, incubated for up to five days at 37°C and tested for the Broth clarity. Results. In presence of the metal concentrations ranged between 50 to 500 mM inhibitory action towards S.pyogenes bacteria was registered as relatively low due to the effects of copper or ferrous ions, as intermediate due to the presence of cobalt, nickel or manganese, and as high due to the effects of zinc ions. At the same time ferrous or copper ions demonstrated high bactericidal activity, zinc ions showed relatively low one, whereas manganese, nickel or cobalt were characterized by the lack of bactericidal action registered. Conclusion. Inhibitory action of heavy metal divalent ions on the lawns of S.pyogenes cultures probably includes bacteriostatic and bactericidal components which impact is determined by primary mechanisms of the ions toxicity.

Aim. To study the effect of OprF and aTox proteins of Pseudomonas aeruginosa on the cytokine profile of mice dendritic cells. Materials and methods. Dendritic cells (DC) were obtained from bone marrow cells of BALB/c mice when cultured with 20 ng/ml of recombinant GM-CSF and IL-4 (Biosource, USA). OprF and aTox of P. aeruginosa were used as the inducer of maturation of DC. The level of cytokines was determined in supernatants of DC using the Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (BioRad, USA). Results. Evaluation of the profile and level of cytokines produced by dendritic cells of mice demonstrates the high activity of mature DC. Under the influence of recombinant proteins OprF+aTox, both large amounts of Th-1 cytokines were synthesized: IL-1a, IL-1P, IL-6, TNF-a, Th-2 cytokines: IL- 4, IL-10, IL-13, regulatory cytokines: IL-12, IFN-y, IL-17A and chemokines: KC (CXCL1), MIP-1a (CCL3), MIP-1e (CCL4), RANTES (CCL5). In our studies, we demonstrated the possibility of obtaining mature dendritic cells from the bone marrow of mice under the influence of a complex of P. aeruginosa antigens. Conclusion. The candidate Pseudomonas aeruginosa vaccine based on its recombinant proteins OprF and aTox induces the production of chemokines and Th-1, Th-2, Th-17 cytokines by mice dendritic cells.

Aim. In this paper we investigate the impacts of co-incubation of Borrelia miyamotoi with neutrophils. Materials and methods. Spirochetes B. miyamotoi, strain HT31, were incubated 3 hours at 37°C with neutrophils of healthy donors (5*10⁶ cells/ml) in a 1:1 ratio. The incubation medium contained also non-immune serum of healthy blood donors (SHD) and, in some experiments, high-immune serum of patients recovered from ITBB-BM (S-ITBB-BM). The proportion of neutrophils that bound borrelia, as well as the number and viability (mobility) of free borrelia, was estimated by dark-field microscopy. Results. Free-swimming borrelia remain viable in SHD or heat-inactivated S-ITBB-BM, but about 10% of borrelia are associated with neutrophils. In S-ITBB-BM with neutrophils, the proportion of viable borrelia among free ones decreases by approximately 10% compared to S-ITBB-BM without neutrophils; in addition about 15% of bor-relia become bound by neutrophils. If chemoattractant fMLP was added, the proportion of neutrophils binding borrelia increases to 25%, and the proportion of immobilized non-bound bor-relia reaches 40%. Conclusion. Although neutrophils are able to destroy borrelia with or without direct contact, under model conditions the combined effect of blood neutrophils and high-immune human serum does not provide 100% elimination of B. miyamotoi.

Aim. To develop an optimal algorithm for laboratory diagnosis of C. difficile associated diarrhea in context of obtaining the most reliable analysis results. Materials and methods. 211 patients with clinically significant C. difficile associated diarrhea participated in the study. Luminal faeces were analysed by immunological (immune chromatographic assay, ICA; ELISA) bacteriological and molecular (GENEexpert rtPCR system, Cepheid, USA) methods. Results. We isolated 126 C. difficile strains from 211 samples of luminal faeces. We identified glutamate dehydrogenase (GDH) in 54% of cases (n=68) by means of immune ICA, and in 11.1% of cases (n=14) by means of ELISA. The increase of bacterial concentration is associated with the growth of sensitivity of GDH detection by immunological tests (p<0.05). We defined a dramatic increase of GDH detection sensitivity starting from 10⁷ CFU/g concentration. Toxins A and B evaluation by immunological tests only leads to false-negative results in 19,8% (ICA) and 55,6% (ELISA) of toxin A positive cases and in 35,7% (ICA) и 44,4% (ELISA) of toxin B positive cases. Conclusion. Three-step diagnostic algorithm for antibiotic-associated diarrhea is characterized by high replicability, sensitivity and specificity. It is based on step-by-step performance of the tests for detection of toxinproducing C. difficile. Application of such approach ensures timely diagnosis, local microbiological monitoring and epidemiological control of C. difficile associated infection.

Aim. The aim of the current study was to analyze the genome structure of the M. bovis BCG-1 (Russia) sub-strain, used for the vaccine production, as well as its genome stability within the entire production process. Materials and methods. Whole genome sequencing and M. bovis BCG-1 (Russia) working seed lot and for the last production passage of the sub-strain cultivation from a number of the vaccine batches. Additionally, VNTR sequences of 24 locus analyses, RD patterns comparison, as well as spoligotyping were performed. Results. The whole genome sequence of the M. bovis BCG-1 (Russia) working seed lot was assembled, annotated and deposited to GenBank. On the basis of DU2- and RD-regions analyzes M. bovis BCG-1 (Russia) sub-strain was confirmed to be belonged to BCG Russia strains of DU2-I group. Whole genome sequencing followed by comparative analysis of RD patterns and SNPs confirmed the stability of the vaccine sub-strain genome from the working seed lot to a number of the vaccine batches obtained within the two-years period. VNTR profile and spoligopattern exactly matched the M. bovis BCG-1 (Russia). Conclusion. Thus the M. bovis BCG-1 (Russia) sub-strain genome identity and stability have been studied and demonstrated. The obtained result confirmed the vaccine production process consistency.

Aim. Influence of the plague agent plasmid content on biofilm formation in vivo and death rate of fleas-vectors with different vector activity in experiment were analyzed. Materials and methods. Three Yersinia pestis strains: virulent I-3230 (pYT, pYV, pYP) and I-2638 (pYT, pYV, pYP, pTP 33), and its selected avirulent isogenic clone I-3480 lacking two plasmids (pYV, pYP) were used. Three species of fleas were artificially infected: 477 individuals of Xenopsylla cheopis (a highly active vector), 441 - Citellophilus tesquorum (an active vector), 519 - Frontopsylla lucu-lenta (a low-active vector). The peculiarities of Y. pestis biofilm formation in fleas were estimated by a portion of individuals with bacterial «conglomerates» and «blocks» for a feeding. Death rate of the insects was defined by the percent of the dead fleas at each feeding. Results. All three flea species infected by Y. pestis strains carrying an additional plasmid pTP33 (I-2638 and I-3480) demonstrated the increase of the individual number with various biofilm forms in comparison with the three-plasmid strain I-3230. In X. cheopis it occurred due to the blocked insects, in C. tesquo-rum - mainly due to the fleas containing «conglomerates», in F. luculenta it was completely connected with ectoparasites with «conglomerates». A share of X. cheopis and C. tesquorum died at a feeding was higher in ectoparasites infected with I-3230 strain and F. luculenta - infected by I-2638. Conclusion. Y. pestis strains possessing an additional replicon pTP33 formed a biofilm in the infected insects more often and larger size than a classical three-plasmid variant. Influence of the strain plasmid content on death rate of the infected fleas depended on a vector species.

Aim. To study the effect of N-acetyl-L-cysteine on biofilm of V. cholerae of different sero-groups isolated from various sources and with different epidemiological significance (the presence/ absence of the ctx AB genes and tcpA). Materials and methods. Bacterial eiiltiire of Vibrio cholerae El Tor O1 and O139 serogroups were grown as biolms. We have estimated the influence of the drug N-acetyl-L-cysteine at a concentration of 0.5 - 4 mg/ml on the formation, of the formed biofilm and in planktonic form. Results. Discovered antibacterial activity of N-acetyl-L-cysteine. Noted that it was influenced as in the formation, of the already formed biofilm and the planktonic form, the representatives of the Vibrio cholerae El Tor O1 and O139 serogroups in concentrations of 2 - 4 mg/ml, showing an antibacterial effect regardless of the presence/absence of genes ctx and tcpA AB. Conclusion. Identified the antibacterial action of the drug N-acetyl-L-cysteine against biofilm of V. cholerae indicates the desirability of considering the possibility of using drug therapy in cases variety of diseases caused by causative agents II - IV groups pathogenicity.

Seroepidemiology is a potentially powerful tool for predicting and monitoring the effectiveness of specific prevention program using studies of antibody prevalence. The availability of a certified collection of blood serum (serum bank) allows to carry out a reliable assessment of population immunity to vaccine-preventable diseases; to determine the degree of epidemiological risk of the infection spread in various areas of the country; to implement short-term and long-term forecasting of changes in the situation on topical infections; to substantiate preventive measures in the system of biosafety for defined population groups and decreed contingents; to provide information necessary for making the optimal management decisions.

Data on the state of the microflora of HIV-infected people and its participation in the progression of the disease are presented. Mechanisms and markers of bacterial translocation through the intestinal mucosa into the bloodstream, their importance for HIV-infected people are described. Study of intestinal microbiome in different groups of HIV-status people controversial data on the phylogenetic diversity of the intestinal microflora are shown. However, in most studies in HIV-infected increase in the intestine of members of the genus Prevotella, reduction of the quantitative level of Bacteroides spp. increase in the proportion of Proteobacteria compared to the other members of the intestinal flora are noted. It is shown that Proteobacteria in HIV-infected patients are more metabolically active than HIV-negative individuals. Further studies of the intestinal microbiome in HIV-infection are presented.

Ebola virus that composed Ebolavirus genus of Filoviridae Family causes severe hemorrhagic fever in humans with high case-fatality rates (up to 90%). The Ebolavirus genus includes Ebola-Zaire, Ebola-Sudan, Ebola-Reston, Ebola-Tai Forest and Ebola-Bundibugyo viruses. The date about epidemic outbreaks of disease, reservoirs of infection, accidental hosts of Ebola virus are presented in this review. The date about natural reservoirs of infection are accessed only for Ebola-Zaire and Ebola-Reston viruses. For Ebola-Sudan, Ebola-Tai Forest and Ebola-Bundibugyo viruses such information is absence. The bats are natural reservoirs for Ebola-Zaire and Ebola-Reston viruses. The formation of natural reservoirs of filoviruses assumes possibilities of existence of several hosts. The interrelation of Ebola virus and their hosts, dynamics of infection are the classical «susceptible-infected-immune» (recovered) cycle. The likely schemes of rises of epidemic outbreaks, caused by Ebola-Zaire virus are suggested.