Biosafety of personnel of microbiological laboratories in the context of the Federal Law of the Russian Federation № 492-FZ of December 30, 2020 «On the biological safety of the Russian Federation¼.

One of the most important requirements for the personnel of microbiological laboratories working with pathogenic and infectious agents is the observance of precautionary measures and the implementation of a set of preventive measures, collectively interpreted as biological safety (biosafety). To a large extent, biosafety problems are also relevant for all clinical laboratories working with biosubstrates, with the potential threat of containing pathogens of bloodborne infections in them. On December 30, 2020, the President of the Russian Federation signed Federal Law № 492 «On the Biological Safety of the Russian Federation¼ (№ 492-FZ), which regulates the basic legal norms and regulation of biosafety issues, as well as a list of measures to prevent the risks of the spread of infections due to accidents, bioterrorist acts and sabotage. The current pandemic of the coronavirus infection COVID-19 has demonstrated, on the one hand, the epidemiological vulnerability of the single world space, and on the other hand, the decisive influence of biological emergencies on the emergence of negative political and economic processes in the world community. In this regard, the issues of ensuring biosafety in the work of microbiological laboratories in the context of protecting personnel and the environment from accidental or unintentional spread of infections are relevant. Working with pathogenic biological agents in microbiological laboratories is constantly associated with the risk of accidents and possible laboratory infection (laboratory-acquired infections) of employees, environmental pollution if the requirements of regulatory documents on biological safety are not met. In accordance with the requirements of № 492-FZ, in order to prevent biological threats, it is necessary to create a system for monitoring biological risks in microbiological laboratories when working with any infected material.

Ultrastructure and Morphological Variability of Non-Culturable Forms of Yersinia pseudotuberculosis Bacteria.

One of the mechanisms underlying the appearance of chronic infections is transition of pathogens into a non-culturable state, which is largely associated with the use of antibiotics. We studied ultrastructure of dormant bacteria Yersinia pseudotuberculosis obtained from the vegetative form of strain 512 by inhibition with kanamycin. On the model of the causative agent of pseudotuberculosis we showed that transition of prokaryotes to a dormant state occurs through apoptosis of bacteria. Fragmentation and condensation of chromatin with the formation of electron-dense fibrils, clumps and large conglomerates characteristic of apoptosis were found in the nucleoid zone of the cytoplasm of inhibited bacterial cells. These results are of great importance for understanding the mechanisms of the existence of pathogens in different conditions, as well as for identifying the causative agents of infectious diseases.

Ultrastructural Changes of Bacteria in Static Cultures of Yersinia pseudotuberculosis under Long Storage under Conditions of Low Temperature.

Electron microscopy study revealed changes in the ultrastructure of bacteria of Yersinia pseudotuberculosis strains characterized by significantly reduced reproductive ability and virulence potential after long-term storage at low temperature of 4-8°C. Most bacterial cells contained dark cytosol with reduced cellular material or empty cytosol, while the cell wall was preserved. The revealed ultrastructural changes in the bacterial cells of the static culture of Y. pseudotuberculosis suggest that storage of strains under low positive temperatures could induce the transition of the majority of bacterial cell population to a dormant, non-cultivated state with a decrease in their virulence. This fact is of great scientific and applied importance in studies of causative agents of saprozoonoses, including pseudotuberculosis, which has the etiopathogenetic background of persistent infection.

Functional State of Mesenchymal Stem Cells upon Exposure to Bioactive Coatings on Titanium Alloys.

Bioactive coatings on implants affect osteogenic differentiation of mesenchymal stem cells (MSC). We studied the morphofunctional state of bone marrow MSC cultured on the surface of calcium phosphate coatings on titanium formed by plasma electrolytic oxidation (PEO). The biocompatible properties of the coatings manifested in the absence of the cytotoxic effect on cells. High expression of receptors (CD90, CD29, and CD106), enhanced synthesis of osteocalcin and osteopontin, and changes in surface architectonics of MSC adherent to the samples confirmed osteoinductive properties of the calcium phosphate PEO coating.

[A comparative evaluation of differential and diagnostic properties of the Serov's agar medium and the base of selective agar for isolating enteropathogenic yersinia.]

The interest in the problem of enterоpathogenic yersinioses in the world is associated with the ubiquitous distribution of these infections and the clinical significance of their pathogenic agents, Yersinia enterocolitica and Y. pseudotuberculosis. In spite of the novel diagnostic tools, microbiological cultivation on nutrient media still remains the main method of their verification. As an agar of choice, American and European practical guidelines recommend a selective agar for Yersinia, which is widely used in many countries for isolating pathogenic Yersinia from clinical material. The authors compare the differential and diagnostic properties of the base of selective agar with those of the Serov's agar medium, as one of the substrates for Yersinia recommended by the current Russian regulatory documents. Based on the studies conducted, a conclusion has been made that the Serov's agar medium is not inferior to its foreign analogue in the differential and diagnostic properties for identification of enteropathogenic Yersinia.

THE STRUCTURAL CHANGES OF MACROPHAGES INFECTED WITH TICK-BORNE ENCEPHALITIS VIRUS.

Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.

Morphogenesis of Experimental Infection Caused by Plasmid Variants of Yersinia pseudotuberculosis.

The dynamics of pathomorphological changes in response to infection with plasmid variants of Yersinia pseudotuberculosis was studied in experimental animals. Variability of cell injuries in pseudotuberculosis histopathology depended on the plasmid-associated virulence of the infection agent. Infection with highly virulent two-plasmid strain pYV48:pVM82 MDa and Y. pseudotuberculosis strain with low virulence with the only plasmid pVM82 MDa led to the development of cell destruction (necrosis and apoptosis) in the target organs. Apoptosis predominated in response to infection by plasmid variant pVM82 MDa with low virulence.

[Pathomorphism of Hantavirus infection under prolonged heat stress]. / Patomorfoz khantavirusnoi infektsii na fone prolongirovannogo teplovogo stressa.

AIM: to provide the morphological characteristics of experimental Hantavirus infection under heat stress conditions to identify the possibilities of its modeling in resistant laboratory animals. MATERIAL AND METHODS: Experiments were carried out on outbred albino mice that were divided into 4 groups: 1) intact mice unexposed to heating; 2) those exposed to heating; 3) Hantavirus-infected animals unexposed to preheating; 4) those exposed to preheating. The animals in Groups 2-4 were long exposed to heat stress at a temperature of 30 °C for 3 hours during 3 days. Strain Aa 60343 (PM-79-95) of the Far East genovariant of Hantaan virus (the genus Hantavirus, family Bunyaviridae) from the collection of the G.P. Somov Research Institute of Epidemiology and Microbiology was used to induce infection. The animals in Groups 3 and 4 were intraperitoneally injected with 700 FFU of Hantavirus per mouse. Materials (lung, liver, kidney, and spleen) taken from Groups 2-4 animals were collected for histological examination on days 1, 3, 7 and 14 of observation. RESULTS: The intact albino mice in Group 1 showed no histopathological changes in the organs. After heat exposure, Group 2 animals were found to have an immunomorphological response in the interstitial tissues of the lung, liver and kidney in partial lymphoid hypoplasia of the spleen. There were no signs of inapparent infection in the presence of marked immunomorphological changes in the organs in Group 3 of hantavirus-infected animals unexposed to preheating. In Group 4, those exposed to preheating exhibited dystrophic and destructive changes in the target organs (lungs, kidneys) in the presence of immunodeficiency manifestations of manifestations that were more pronounced in dead animals. CONCLUSION: In an unresponsive model (adult albino mice), Hantavirus infection caused only obvious immunomorphological changes in the organs. Prolonged preheat stress in the hantavirus-infected animals promoted inapparent infection and morphological manifestations of induced secondary immunodeficiency that was responsible for the manifestation of an infectious process in some animals.

[PLASMID-ASSOCIATED VIRULENCE OF YERSINIA PSEUDOTUBERCULOSIS AND INFECTIOUS PROCESS].

Literature data regarding genetically-determined pathogenicity factors of Y pseudotuberculo- sis and associated manifestations of this infection caused by various plasmid types of the causative agent are generalized. Principal attention is given to features of cell-tissue alterations mediated by virulence plasmid pYV, as well as effects of pathogenicity of an understudied pVM82 plasmid present only in Y pseudotuberculosis sttains causing clinical-epidemic manifestation of the infec- tions as Far East scarlet-like fever (FESLF). The data obtained on the ability of far-eastern strains to produceYPMa super-antigenj Ypseudotuberculosis-derivative mitogenA, probablygive evidence on its key role in FESLF pathogenesis. Variability of damage of innate immunity cells and target- organs caused by various plasmid types of Y pseudotuberculosis by virulence could determine polymorphism of clinical-morphological manifestations of this infection. In-depth understanding of dependency of immune pathogenesis mechanisms of the disease on molecular characteristics of the causative agent opens up-perspectives of enhancement of diagnostics and prognosis of the severity of the course of pseudotuberculosis and yersiniosis in human in general.

[The antimicrobial strategies of neutrophils under infection pathology.]

The neutrophilic granulocytes were traditionally considered exclusively as phagocytes - killer cells of microorganisms invaded human organism. The discoveries of last decade permitted to significantly reconsider this role and importance of neutrophils in implementation of affect mechanisms of inherent and adaptive immunity. The modern achievements expanded our conceptions about anti-microbial strategies of neutrophilic granulocytes under infection pathology: phagocytosis, degranulation and development of neutrophil extracellular traps. These strategies also play a key role in damage of tissues, providing cytotoxic functions. The article presents actual data concerning the role of interaction between neutrophils and adaptive immune cells in development of mechanism of destructive pathological activation of immune response that results in an auto-aggression, induction of chronic inflammation and development of oncologic and auto-immune diseases.

[The metabolism of the innate immunity cells in bacterial infections].

Metabolic activity of innate immunity cells infected by various doses of Gram-negative (Yersinia pseudotuberculosis, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus, Listeria monocytogenes) bacteria has been investigated. Using various animal models we found that during the initial period (up to 2 days) changes of infection in cellular responses depend on the type of the pathogen. In response to infection caused by Gram-negative bacteria predominant neutrophil accumulation in the foci of inflammation was observed, while Gram-positive bacteria induced preferential accumulation of macrophages. The study of metabolism of these cells showed that the response of terminally differentiated primed phagocytes to pathogen appearance was higher than in cells circulating in blood. In addition to the priming state the phagocyte reactivity is influenced by the bacterial load. At a low phagocyte/microbe ratio the cells reaction is almost undetectable, while an excess of microorganisms causes (despite of the increase of the phagocytic parameters) the hyperactivation of cell metabolism and production of maximal amounts of bactericide agents, which exhibit a damaging effect on the cell itself.

Effects of immunomodulators on functional activity of innate immunity cells infected with Streptococcus pneumoniae.

Low activity of bactericidal enzymes was found in innate immunity cells infected with S. pneumonia. The death of these cells was fastened under these conditions. On the contrary, treatment with antibiotic maxifloxacin was followed by an increase in activity of bactericidal enzymes in phagocytes and induced their death via necrosis. Analysis of the therapeutic properties of immunomodulators tinrostim and licopid in combination with maxifloxacin showed that these combinations correct functional activity of cells infected with S. pneumonia.

Effect of thermolabile toxin from Yersinia pseudotuberculosis on functions of innate immunity cells.

The thermolabile toxin of Yersinia pseudotuberculosis produces a selective dose-dependent stimulating effect on functional activity of innate immunity cells. Prolonged apoptosis-inducing action of the toxin was associated with activation of enzymes of the oxygen-dependent system (LDH and myeloperoxidase) at the early terms of observation (up to 3 h). In turn, increased number of macrophages with apoptotic changes was noted at the early stages of contact with the thermolabile toxin (5 h), and its further growth was observed against the background of activation of mitochondrial enzymes and production of NO metabolites.

Pathomorphosis of experimental infection in mice, infected by Streptococcus pneumoniae, under the effect of immunotropic drugs.

Pathomorphological changes in the organs of animals intranasally infected with Streptococcus pneumoniae were studied under conditions of immunotropic therapy added to antibiotic therapy. The pathomorphosis in the lungs, spleen, and thymus in animals treated with likopid, tinrostim, and roncoleukin was described. A positive time course of the pathological process in experimental animals in comparison with intact animals and animals receiving no immunotropic drugs was demonstrated.

[The new views to pseudotuberculosis pathology].

For the first time the nature of cell damages under pseudotuberculosis as a generalized infection has been analyzed by positions of the modern knowledge about types of the cell death. The own and literature data have pointed to presence the apoptosis-induced effect of Yersinia pseudotuberculosis, a causative agent of this infection. In conclusion, the typical pathological changes for pseudotuberculosis infection such as granuloma formation with the central karyorrhexis could be appearance of apoptotic and secondary necrotic changes.

[The metabolic activity of neutrophils and monocytes as the model for the study of the organism biocompatibility with different materials].

In stimulated status neutrophils and monocytes in the foci of introduction the different materials are capable to excrete of biologically active substances by means of which if influence on processes of reparation. The express changes of blood leukocytes activity in response on the introduction of stimulated agents are assume as a basis of the proposed model of the organism biocompatibility with different materials. On example of the influence of different materials (ceramics, titanium, latex, surgical steel and copper), from which can be made implant and surgical instruments it was showed that the leukocytes were showed the different reaction of enzyme systems. This model allows the completely and objectively to study the influence of different materials on the neutrophyls and monocytes--the effected cells of the inflammatory process. Moreover, the activity of cation proteins and plasma membrane enzymes of leukocytes is the most expressed factor.

Pathogenetic role of Yersinia pseudotuberculosis endotoxin in hemostasis and microcirculation disturbances.

We studied the role of Y. pseudotuberculosis endotoxin (LPS) in the pathogenesis of hemostasis and microcirculation disorders. It was found that changes in the hemostasis system after injection of LPS had biphasic character corresponding to the stages of DIC syndrome development. Pathomorphological findings in animals with endotoxemia induced by Y. pseudotuberculosis LPS attested to increased permeability and destruction of the vascular endothelium in the microcirculatory bed and focal degenerative and necrotic changes in cells of the target organs (kidney, liver, and lungs) progressing with increasing the duration of the pathological process, which was determined by microcirculation disturbances and development of multiple organ failure.

[Comparative characteristics of oxygen-dependent and nitroxide-forming enzymatic systems of macrophages in Staphylococcus aureus and Listeria monocytogenes infections].

The reactivity of oxide-dependent and nitroxide-forming enzymatic systems of macrophages infected by bacteria, characterized by intraphagosomal (Staphylococcus aureus) and intracytoplasmatic (Listeria monocytogenes) localization in the phagocyte has been investigated. The correlation analysis revealed links between indices of the activity of oxide-dependent and nitroxide-forming enzymatic systems of infected macrophages.

Changes in the metabolic activity of macrophages under the influence of tick-borne encephalitis virus.

The metabolic activity of macrophages infected with tick-borne encephalitis virus (TBEV) affecting the human nervous system has been studied for the first time. The penetration and reproduction of TBEV in the macrophages stimulated their oxygen metabolism, increasing the activity of NADPH-oxidase complex, as well as the mitochondrial enzymes lactate dehydrogenase, succinate dehydrogenase, and cytochrome oxidase. A wave-like change in the activity of these enzymes in the macrophages reflected the reaction of the cells to the penetration of the virus in the first period (within 3 h) and to the synthesis of the virus particles and their exit into the extracellular space in the second period (from 5 to 48 h). In the macrophages infected with TBEV, accumulation of NO metabolites was observed. In the late period of the examination (1-4 days), the activities of superoxide dismutase and lysosomal enzymes (nonspecific esterase and acid phosphatase) were detected. Thus, the early increase in the activity of the cell enzymes indicates the activation of the macrophages, and the subsequent increase in their activity corresponds to the enhanced synthetic activity of the macrophages.

[Functional activity of mouse peritoneal macrophages contaminated with gram-positive bacteria].

The analysis of the functional and enzymatic activity of mouse peritoneal macrophages contaminated with Staphylococcus aureus and Listeria monocytogenes virulent strains is presented. The low bactericidal and digestive activity of these cells with respect to the above-mentioned microorganisms was determined. In this study a decrease in the activity of plasmatic membrane enzymes (5'-nucleotidase and ATP-ase) of macrophages contaminated with S. aureus and L. monocytogenes was observed, which was indicative of the stimulation of phagocytes. A rise in the activity of the oxygen-dependent system of macrophages contaminated with S. aureus and L. monocytogenes was detected by means of the nitro blue tetrazolium test. At the same time a decrease in the intracellular content of nitrogen oxide end metabolites in macrophages was detected with a rise in content of nitrogen oxide in the supernatants.