Influence of monostrain and multistrain probiotics on immunity, intestinal ultrastructure and microbiota in experimental dysbiosis.

The biological effects of three probiotic strains Lactobacillus rhamnosus K32, Bifidobacterium longum GT15, Enterococcus faecium L3 and their mixture were studied using a model of dysbiosis induced in rats by antibiotics. It was found that after taking different probiotics intestinal microbiota changed in a strain-specific manner. The maximal activity against pathogens was revealed after the administration of a mixture of bacterial strains under study or a single strain of enterococci. The strain E. faecium L3 showed the most activity against both Klebsiella spp. and Bacteroides fragilis. It helped to restore the original content of Faecalibacterium prausnitzii. The number of Klebsiella spp. was the same in the group receiving L. rhamnosus K32 and the group of animals, which was not consuming probiotics. Different probiotic strains included in the composition had various immunological effects. Probiotic bifidobacteria, enterococci and the mixture of three probiotics stimulated of mRNA expression of interleukin (IL)-10 in mesenteric lymph nodes. The changes in microbiota after consuming an enterococcal probiotic correlated with an increase in transforming growth factor (TGF)-ß and IL-10 content in blood serum and an increase of the intestinal mucus layer. Consumption of L. rhamnosus K32 led to the stimulation of IL-8 expression in mesenteric lymph nodes. Control group not receiving probiotics was characterised by expression of pro-inflammatory cytokines, damage of epithelial cells and the destruction of their tight junctions. The damage to the ultrastructure of the mucosa was prevented in all the groups taking probiotics.

[COMPARATIVE ANALYSIS OF TIGHT JUNCTIONS OF EPITHELIUM OF RATS JEJUNUM UNDER THE EFFECT OF LIPOPOLYSACCHARIDE AND CHOLERA TOXIN].

AIM: Comparative study of tight junctions and ultrastructure alterations of enterocytes of mucous membranes of jejunum of rats under the effect of lipopolysaccharides and cholera toxin. MATERIALS AND METHODS: Lipopolysaccharides (Sigma-Aldrich, Germany) and cholera toxin (Sigma-Aldrich, Germany) were used. The study was carried out in Wistar line rats. Effect of lipopolysaccharides and cholera toxin on epitheliocytes was carried out by a method of withdrawal of segments of rat jejunum and their incubation with the specified substances. Comparative analysis of ultrathin sections of enterocytes of jejunum of rats and tight junctions between them was carried out in control and under the effect of lipopolysaccharides and cholera toxin. RESULTS: Effect of lipopolysaccharides on ultrastructure of enterocytes of rat jejunum manifested in the change of cell form as a result of increase of intercellular space without destruction of tight junctions. Disappearance of desmosomes, increase of nuclei and more pronounced ER were noted in some epitheliocytes. Effect of cholerogen on epitheliocytes of mucous membrane of rat jejunum by a number of signs is similar to the effect of lipopolysaccharides, that manifested in an alteration of ultrastructure of cell, the form of those also transformed as a result of an increase of intercellular space, this process was not accompanied by destruction of tight junctions. Disappearance of folding of the lateral region of plasmatic membrane of cells and a reduction of a number of microvilli was observed under the effect of cholera toxin. CONCLUSION: A similar character of effect of lipopolysaccharides and cholera toxins on ultrastructure of cells and region of tight junctions of enterocytes of rat jejunum was detected, both substances caused an increase of intercellular space without the destruction of tight junctions.

[FEATURES OF FORMATION OF BACTERIAL BIOFILMS IN CONDITIONS OF SPACE FLIGHT].

AIM: Study the effect of microgravitation on the formation of Lactobacillusplantarum 8PA-3 bacterial biofilms in the conditions of space flight. MATERIALS AND METHODS: Information on the ef- fect of microgravitation on the development of biofilms was obtained during study of L. plantarum 8PA-3 probiotic lactobacilli in special equipment in the process of execution of space experiments in the Russian segment of the International Space Station. Comparative analysis of growth of plankton and biofilm forms of cells developing in the conditions of space flight and surface conditions was carried out by microbiologic and electron-microscopy methods using scanning and transmission electron microscopy. RESULTS: Accelerated dynamics of formation of L. plantarum 8PA-3 lactobacilli biofim on the surface polymer substrate was shown for the first time during the space experiment. Microbiological anfalysis of the bacterial culture has also confirmed the accelerated growth of L.plantarum 8PA-3 under microgravitation compared with surface conditions. Ultrastructure of plankton form of L. plantarum 8PA-3 taking part in formation of biofilms inconditions ofmicrogravitationwas detected for the first time inthe space experiment. CONCLUSION: Data on comparative electron-microscopic analysis obtained in space experiments are important for scientific justification of the effect of icrogravitation on bacterial communities developig as biofilms - the most natural form of existence of microorganisms. The results obtained could be taken into consideration during creation of novel antibacterial means and disinfectants as well as methods of treatment of surfaces of modules of piloted space complexes that could allow to clarify methods of effective prophylaxis of biofilm spread which pose a risk of health of the crew and normal functioning of equipment in the International Space Station.

[Analysis of prophylactic and therapeutic effect of probiotic preparations from position of new scientific technologies].

In this review new scientific technologies (genomics, proteomics, metabolomics, transcrip- tomics) were used to evaluate the prophylactic and therapeutic action of probiotics, which are a major component ofthe normal human microflora (microbiota). Modern terms, definitions, classification of probiotic preparations are provided in the paper, the list of the probiotics registered in the Russian Federation is also submitted. The review analyzes the majority of mechanisms of probiotics action on a human body. The problem of safe application of probiotics is considered along with the detailed characteristic of the most effective production probiotic strains. New scientific technology to assess the effects of probiotic bacteria on the various functions of the macroorganism are also examined. In the review the special attention is paid to discussion of effectiveness of the probiotics impact in chronic infectious and metabolic disease processes (atherosclerosis, lipid distress syndrome, type 2 diabetes, obesity, etc.), which are the most active during dysbacteriosis and the destruction of normal microflora. From data of this article clearly that new scientific technologies will allow us to establish the functions of proteins that regulate metabolic and signaling pathways and affect the expression of genes required for the adaptation of probiotic strains in contact with the human body. In this review it is shown that the successful solution of this problem is closely connected with application of new scientific technologies for studying the composition and functions of the human microbiota, methods of active influence on her, and also with development of more sophisticated and effective probiotic preparations.

[EFFECT OF PULSE-PERIODIC CORONA DISCHARGE ON VIABILITY OF ESCHERICHIA COLI M17 CELLS IN BIOFILMS].

AIM: Detection of bactericidal effect of pulse-periodic corona discharge (PPCD) on cells and biofilms of Escherichia coli M17. MATERIALS AND METHODS: A gas-discharge device was created based on PPCD in air with power supply parameters: amplitude values of voltage of 30 - 60 kV, pulse repetition rate of 250 - 400 kHz. Ultrastructure changes in cells and biofilms of E. coli M17, affected by PPCD, generated in air, were studied by typical methods of transmission electron microscopy. RESULTS: Disturbances of integrity of surface and abyssal structures of biofilms, as well as changes of morphological properties of E. coli M17 cells, characteristic for sub-lethal heat impact, were detected. Destructive changes of bacterial cells were developed by formation of focal disturbance of cytoplasmic membrane, extension of periplasmic space, formation of globular structures, characteristic for heat effect, and destruction of cytoplasm. CONCLUSION: Bactericidal effect of PPCD on E. coli M17 cells as part of biofilms was shown. Destructive morphological changes in cells and biofilms of E. coli M17 after the effect of PPCD were detected for the first time on electron-microscopic level.

[Ultrastructure of microbial biofilms during intercellular interactions of bacteria in communities].

Analysis of summarized data obtained by us on ultrastructure of microbial biofilms of opportunistic bacteria is presented. A complex ultrathin organization of lactobacilli, enterobacteria, staphylococci and enterococci biofilms discovered during electron microscopy is described. The presence of surface film and polysaccharide matrix that determine increased resistance of intrabiofilm bacteria against the effect of protective immune factors of the organism and etiotropic preparations is demonstrated in all the studied comminutes. A varied response of bacterial cells contained in the biofilm during antagonistic effect of symbiont probiotic bacteria was discovered.

Ultrastructure of colony-like communities of bacteria.

Colony-like communities are poorly studied forms of bacterial growth on agar. These communities are formed after the growth of large amounts of bacteria simultaneously plated onto a limited area of agar, while "classical" colonies are formed as a result of single bacterial cell multiplication. Colony-like communities of Gram-negative and Gram-positive bacteria differ from "classical" microbial colonies in their ultrastructural organization. Almost every cell in colony-like communities has an individual capsule-like envelope (glycocalyx). The cells in these communities are characterized by accelerated ageing. In the course of their development both bacterial colony-like communities and "classical" colonies produce a film, the basic part of which is represented by an elementary membrane. In contrast to "classical" colonies, the thickness of the amorphous layers of this film in colony-like communities did not significantly increase after 24 h of cultivation. The formation of a three-dimensional network of cells in colony-like communities is similar to this process in "classical" colonies. The intercellular matrix of colony-like communities contains numerous membrane vesicles, and has a more amorphous structure and higher electron density than that of "classical" bacterial colonies.

Ultrastructure of the surface film of bacterial colonies.

The structure of the surface of colonies of various Gram-negative and Gram-positive bacteria was examined by transmission electron microscopy. The results indicate that bacterial colonies in the course of their development produce a film which becomes thicker with increased duration of growth. The basic part of the film is an elementary membrane, which is a stable structure with a large surface area. The inner and outer surfaces of the film membrane are covered by amorphous layers. These layers are thicker in the surface film of Gram-negative bacterial colonies than in those of Gram-positive bacteria. Membrane vesicles from the bacterial colonies take part in the formation of the surface film. The presence of the film on the surface of the colonies of different bacteria suggests that this structure may play an important role.

Surface films of Escherichia coli colonies.

Escherichia coli colony surfaces were examined using SEM and TEM. The results indicated that bacterial colonies in the course of their development produce surface films which become thicker with increased growth duration. Membrane vesicles contribute to the formation of the surface film. The complex organization of the film suggests that it may perform specific functions.

Morpho-physiologic characteristics of Escherichia coli and Shigella flexneri 2a carrying the htpR15 gene.

Electron microscopic examination of the morphology of normal Escherichia coli MC4100 and Shigella flexneri cells and their htpR-defective recombinants has shown the presence in the latter of noncrystalline granules after growth at 37 degrees C. The defect of the htpR gene leads to some changes in ultrastructure of bacterial colonies and the intensification of the processes of development and aging of colonies as integral structures. These phenomena are manifested by early formation of intercellular contacts and thick surface films as well as the enhanced destruction of microorganisms. The recombinant Shigella strain grown at 30 degrees C retained its ability to provoke keratoconjunctivitis.

Ultrastructural features of microbial colony organization.

The ultrastructure of microbial colonies was studied. Inside the colonies three types of intercellular contacts were demonstrated. In the colonies of Gram-negative bacteria, the cells were found to be connected by tight adhesions of outer membranes of the cell walls and membrane bridges. In the colonies of Gram-positive bacteria, the intercellular contacts were formed by fusion of peptidoglucan layers of the cell walls. Bacterial cells were differentiated by the presence of a capsule-like envelope. The obtained data indicate the existence of elements of cellular cooperation and specialization in microbial colonies.