[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.

[Commensal microflora and endogenous inductors of pathophysiological reactions of innate immunity].

This paper content analytical review of literature on commensal microflora and endogenous inductors of pathophysiological reactions of innate immunity. The important role of participation in the homeostasis of the intestinal microflora, the repair of tissue damage, maintaining the innate immune system in a state of physiological tone by interacting with receptors pattern recognition receptors. Effects of the interaction of components commensal flora and innate immune receptors play an important role in physiological processes of microorganism in maintaining the balance of the synthesis of pro-inflammatory and anti-inflammatory cytokines and antimicrobial substances.

[ENTEROTOXIGENICITY OF STAPHYLOCOCCUS AUREUS STRAINS, ISOLATED FROM BREAST MILK OF WOMEN, FEEDING CHILDREN WITH INFECTIOUS PATHOLOGY].

AIM: Determination of enterotoxigenicity and ability to synthesize TSST-1 in S. aureus strains, isolated from breast milk of women, feeding children with infectious pathology. MATERIALS AND METHODS: 35 S. aureus strains, isolated from breast milk of women feeding children with varying infectious pathology in hospitals and as outpatients were studied for the presence of staphylococci enterotoxins (SE) of types A and B and toxic shock syndrome toxin (TSST-1). Determination of SEA, SEB and TSST-1 was carried out by enzyme immunoassay. RESULTS: Toxins were detected in 94.2% of S. aureus strains. SEB was synthesized by 86.7%, SEA--34.3%, TSST-1--42.8% of S. aureus strains. Toxins were detected with equal frequencies in healthy women and women with inflammatory diseases of breasts. Differences in frequency of colonization of intestines of children receiving breast milk, infected with toxigenic and non-toxigenic staphylococci strains was not detected. CONCLUSION: A high frequency of occurrence of enterotoxins and TSST-1 in S. aureus, isolated from breast milk of the mother during infectious pathology in the child was discovered. Enterotoxigenic strains can be detected in breast milk in healthy women. Study of the role of breast milk, infected with S. aureus, producing SEA, SEB And TSST-1 in development of child pathology is necessary.

[Intestinal microflora, obesity and type 2 diabetes].

The review of data of the literature on a role of intestinal microflora, genetic features of a macroorganism, exogenic factors and character of a food is presented at obesity and a type 2 diabetes. Researches establish, that development in experimental animals of the induced obesity and the type 2 diabetes, depends on a diet and presence of intestinal microflora. The factors increasing permeability mucous intestines, promote a translocation of intestinal automicroflora and its toxins into macroorganism and a system blood-circulation. Long introduction LPS (endotoxin) of gram-negative bacteria to the special laboratory animals led to development of inflammatory reaction, adiposity and resistance to insulin. The specified phenomena did not develop at LPS introduction to the animals, who have lost receptor CD14 which is necessary for linkage and endotoxin action. Data about change of intestinal microflora and a role of immune infringements are discussed at obesity and the type 2 diabetes occurring into background of low-grade chronic inflammation and metabolic disorders.

[Frequency of occurrence of ivyC and pliC lysozyme inhibitors genes in clinical klebsiella strains].

AIM: Chromosomal ivyC and plasmid pliC lysozyme inhibitor genes frequency of occurrence detection in klebsiella strains of various origins. MATERIALS AND METHODS: 129 strains were studied including K. pneumoniae (n= 115) and K. oxytoca (n=14). Klebsiella strains were divided into 2 groups: group 1 - 62 strains (39 isolated in Kazan and 23 strains isolated from children with klebsiella infection in Moscow). Kazan strains were presented by isolates from young children receiving in-patient treatment regarding klebsiella (n=8), respiratory (n= 17), rotavirus (n=5) and purulent-inflammation infection ofnewborns (n=9). Group 2 (n=67) was composed of strains isolated from children that had received outpatient treatment regarding intestine dysbacteriosis in Kazan (n=37) and Moscow (n=30). ivyC and pliC gene detection was carried out by PCR (N.B. Perunova et al., 2012). RESULTS: Chromosomal ivyC gene determinants were isolated in 40.3% of cases (52 strains of 129), plasmid pliC gene - 6.9% (9 of 129). ivyC gene frequency of occurrence in klebsiella strains isolated from 39 ill children of Kazan was 46.6% of cases (18 of 39), in Moscow clinical strains - 95.5% (22 of 23) cases, wherein 6 strains had ivyC and pliC genes at the same time. Dysbiosis intestine strains had chromosomal ivyC gene at the frequency of 61.2% (41 of 67), 1 strain additionally had pliC gene. CONCLUSION: . The presence of ivyC and pliC lysozyme inhibitor genes in klebsiella clinical strains provides bacteria with selective advantages in various biotopes in the process of bacterial colonization of mucous membrane.

[Bacterial lipopolsaccharides in pathogenesis of gynecological diseases and obstetric complications].

Analysis of literature data and author studies on the role of lipopolysaccharides (endotoxin) of Gram negative bacteria in women genital tract pathology and obstetric complications is presented. The role of endogenous infection associated with altered microecology of intestinal tract and vaginal biotopes of women in the development of endotoxinemia is discussed. The participation of endotoxin in embryo resorption, delay of intrauterine development and antenatal death of fetus, premature birth, pre-eclampsia, placental dysfunction is examined. The level of endotoxinema and pro-inflammatory cytokines is a marker of chronic endogenous infectious-inflammatory disease of various parts of genital tract with damage of a network of female reproduction system organs.

[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.

[Perspectives of disodium salt 2.4-di(1-metoxyethyl)-deuteroporphyrin - IX ("Dimegin") application for photodynamic therapy in non-oncologic cases].

Effects of disodium salt 2,4-di(1-metoxyethyl)-deuteroporphyrine-IX (Dimegin) and the light from Soret band (¼395-405 nm) at the viability of microbial cells and at their potential to form microbial biofilms have been compared with traditional antiseptics. Irradiation of microbial cells of S. aureus, E. coli, C. albicans and others with diode light (power density 0.05 Wt/cm2) caused a bactericidial effect similar to that obtained with standard anticeptics (chlorhexidine and dioxidine). A comparative study of the effectiveness of Dimegin and Photoditazine (a soluble salt of chlorine e6) as photosensitizers have been performed using the test system of erythrocyte hemolysis in vitro under irradiation with light from the Sore band. Results have shown insignificant difference in the photodynamic effect with similar doses of absorbed light and preparation concentration.

[Antimicrobial peptides of lactobacilli].

Data on antimicrobial peptides (AMP) of lactobacilli, mechanism of their damagin effect, chemical nature and genetic control are presented. Regardless of the source of isolation AMP of lactobacilli except reuterin are peptides with low molecular weight (4 - 6 kDa), differ from each other by chemical structure, sensitivity to temperature, effect of various enzymes, active at neutral or more frequently low pH. Especially important are mechanisms ensuring fine regulation of phenotypic expression of bacteriocin synthesis and formation of immunity against their effect. Activity and most importantly the level of their production depend on the conditions in which these bacteria are present and are controlled by a three-component regulation system. This system includes signal peptide (pheromone), sensory histidine kinase, regulator protein activating transcription. Resistance of the producer to the effect of its own bacteriocin is ensured by the so called immunity protein. AMP of lactobacilli are able to influence septoformation, peptidoglycan and protein synthesis, affect cytoplasmic membranes causing their destabilization. Stages of this damaging effect are described: interaction of effector peptides with the membrane of the sensitive cell, positioning of the peptide in the region of connection with protein receptor, submerging into the core of the spiral structure membranes with the formation of a pore and exhaustion of ATP pool leading to cell death. Protection from AMP is determined by specific proteins blocking pore formation in the membrane by direct binding of damaging molecules or their receptors. Perspectives of further studies of the role of antimicrobial peptides of lactobacilli are discussed.

[Intestinal-brain axis. Neuronal and immune-inflammatory mechanisms of brain and intestine pathology].

Mutually directed connections between intestine and brain are implemented by endocrine, neural and immune systems and nonspecific natural immunity. Intestine micro flora as an active participant of intestine-brain axis not only influences intestine functions but also stimulates the development of CNS in perinatal period and interacts with higher nervous centers causing depression and cognitive disorders in pathology. A special role belongs to intestine microglia. Apart from mechanic (protective) and trophic functions for intestine neurons, glia implements neurotransmitter, immunologic, barrier and motoric functions in the intestine. An interconnection between intestine barrier function and hematoencephalic barrier regulation exists. Chronic endotoxinemia as a result of intestine barrier dysfunction forms sustained inflammation state in periventricular zone of the brain with consequent destabilization of hematoencephalic barriers and spread oF inflammation to other parts of the brain resulting in neurodegradation development.

[Application of multiplex PCR for analysis of polycomponent probiotic preparations].

AIM: Development of multiplex PCR for indication and differential analysis of CFU in a probiotic preparation containing 3 various species ofbifidobacteria (Bifidobacterium bifidum, B. longum, B. adolescentis) and 2 species of lactobacilli (Lactobacillus acidophilus, L. plantarum). MATERIALS AND METHODS; Multiplex PCR with primers to 16S RNA genes of 5 species of bacteria was applied. Samples of bacterial colonies were removed from solid nutrient medium and transferred into test-tube for multiplex PCR execution. Bacterial lysates were used as target-matrix for amplification and determination of presence of the required amplicons. RESULTS: Multiplex PCR for detection of CFU of each of the bifidobacteria and lactobacilli strains in samples of 5-component probiotic with accuracy up to species was developed. A reagent kit was fine-tuned and high reproducibility and specificity of the multiplex PCR method with the accuracy of 90 - 95% of cases was shown. CONCLUSION: The multiplex PCR method developed is recommended to be applied for CFU control of each bacterial component in a complex probiotic preparation.

[Systemic endotoxinemia as a pathogenetic factor of pregnancy complication].

AIM: Evaluation of the role of hemocirculation of a bacterial endotoxin in pregnancy complicated with gestosis. MATERIALS AND METHODS: A complex clinical-laboratory examination of 74 pregnant women at the II-III trimester of gestational period including determination of serum cytokine levels (TNFalpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFNgamma), C-reactive protein, LPS, LPS-binding protein and IgG against R-core LPS in blood plasma was performed. RESULTS: In pregnant women with gestosis an increase in endotoxinemia level (p < 0.0001), twofold increase in IgG titers against R-core LPS (p < 0.01) and an increase in LBP concentration by 14% (p < 0.05) without an increase in concentration of C-reactive protein were shown. Cytokine profile in gestosis was characterized by a twofold statistically significant increase of IL-1beta (p < 0.0001), IL-8 (p < 0.0001) pro-inflammatory cytokine levels, a tendency of IL-6 (p = 0.063), TNFalpha (p = 0.13) increase and a decrease of Th1 cytokine concentrations: IL-2 - by 6.5 times (p < 0.0001), IFNgamma--by 2 times (p < 0.0001). CONCLUSION: The results allow to consider gestoses as a manifestation of increased endotoxin translocation into systemic blood flow during gestation, that determines the necessity to improve therapeutic measures in this pathology.

[Presence of genes of genotoxin associated with pks pathogenicity island in Escherichia coli M-17 probiotic strain].

AIM: Study the presence of genetic determinants of pks pathogenicity island containing clb (colibactin) genes in bacteria of the E. coli M-17 production strain by using PCR. MATERIALS AND METHODS: E. coli M-17 cultures isolated from biopreparations bificol (Microgen) and colibacterin (Biomed) and control strain obtained from Tarasevich State Institute on Standardization and Control (Moscow) were studied. Detection of genetic markers of colibacterin was performed by using multiplex PCR with 4 pairs of primers amplifying the main clb genes: clbB, clbN, clbA and clbQ, generating 575, 711, 981 and 820 bp amplicons, respectively. RESULTS: In genome of all the studied E. coli M-17 strains clbB, clbN, clbA and clbQ genes associated with the formation of genotoxic colibacterin were detected. CONCLUSION: Genome of E. coli M-17 bacteria used for production of probiotic preparations colibacterin and bificol contains genetic determinants ofgenotoxin that require further studies in terms of evidence of harmlessness of production bacteria.

[Suppression of viability of staphylococci cells by 1270 nm laser beam].

AIM: Study the effect of 1270 nm wavelength laser exposure on the viability of Staphylococcus aureus and S. epidermidis cells in vitro. MATERIALS AND METHODS: Reference strain S. aureus 209P and clinical isolate S. epidermidis 26/193 that form bacterial biofilm were used in the study. Ica gene presence in S. epidermidis 26/193 that controls biofilm formation was confirmed in the study by PCR with a primer that generates a specific 814 n.p. amplicon. Experimental device by "New surgical technologies, Ltd." was used as a source of emission. The device has a continuous emission mode of laser semiconductor diodes with 1270 nm wavelength. Maximum regulated power of the emission is up to 3 W. Emission power in the studies performed was 150 mW. The time of exposition was 5, 10, 15 and 30 minutes. The amount of viable cells in the experiment and control was determined by calculating CFU/ml. Evaluation of bacterial biofilm formation was performed by the method described in O'Toole G.A. et al. (2000). RESULTS: A decrease of the number of viable forms of S. aureus 209P by a mean of 52 +/- 6.0% and 76 +/- 4.0%, and of S. epidermidis 26/193 by a mean of 48 +/- 4% and 64 +/- 5% for 15 and 30 min exposition, respectively, and a significant suppression of biofilm formation by staphylococci was shown. CONCLUSION: Exposure to 1270 nm laser renders a moderate bactericidal effect on staphylococci cells and significantly suppresses their bacterial biofilm formation.

[Persistence of biologically active components of gastrointestinal tract microflora].

The article is dedicated to examination and analysis of materials on translocation of microflora and its products from intestine to the internal environment of the macroorganism and persistence of biologically active substances of microflora in the bloodstream. High frequency of translocation and persistence of intestine microflora and its components in system bloodstream is shown. Persistent biologically active substances of endogenous microorganisms take part in human physiology and pathology.

[Refinement of taxonomic position of Lactobacillus genus probiotic strains by 16S rDNA and rpoA gene sequencing].

AIM: Revision of the species identification of collection lactobacilli strains based on 16S rDNA and rpoA gene sequencing. MATERIALS AND METHODS: 52 lactobacilli cultures that present mostly Gamaleya Research Institute of Epidemiology and Microbiology (GIMC) collection were studied. 16S rDNA gene fragments were amplified by using Lb16a, Lb16b, 16S-midford, 16S-midrev primers. 2 different reverse primers were used for the analysis of rpoA gene depending on lactobacilli species. DNA fragments sequencing was performed with 3130 Genetic Analyzer (Applied Biosystems/Hitachi) with primers used for amplification. RESULTS: The effectiveness of sequencing of 2 targets for differentiation of species within lactobacilli phylogenetic groups was shown. Species diversity was demonstrated for GIMC lactobacilli strain collection that includes members of 9 species. All the strains marked previously as L. acidophilus were determined to belong to L. helveticus. Strains belonging to recently discovered L. farraginis species that has promising application in agriculture were detected. CONCLUSION: Genetic passports of original strains of 9 species of lactobacilli that are promising for further research.

[Recognition of commensal microflora by pattern recognition receptors in human physiology and pathology].

Contemporary data on the interaction of commensal microflora and Toll-like pattern recognition receptors are presented. These receptors recognize normal intestine microflora in physiological conditions, and this interaction is necessary for the maintenance of homeostasis and damage reparation of the intestine, for the induction of heat shock cytoprotective proteins. As a side effect in disruption of immunologic tolerance and misbalance of protective immunological mechanisms, multiorgan pathologic changes of organs and tissues may develop, including chronic inflammation processes of various localization.

[Destruction of lactobacilli by Staphylococcus aureus bacteriocin-like inhibitory substances].

AIM: Determination of morpho-physiological changes in cells of a lactobacillus industry strain suppressed by antagonistically active clinical cultures of Staphylococcus aureus. MATERIALS AND METHODS: 5 clinical strains of Staphylococcus aureus detected among 42 strains isolated previously in atopic dermatitis were used. Bacteria of the industry strain Lactobacillus plantarum 8PA-3 that synthesize bacteriocin-like inhibitory substances (BLIS) were used as target-cells. Suppression of growth of lactobacilli cells by staphylococci was performed on modified agar nutrient medium MRS by delayed antagonism method. Ultrastructure changes in lactobacilli cells exposed to staphylococci BLIS were studied by various methods of transmission electron microscopy. RESULTS: Destructive changes (exfoliation of small layered fragments of peptidoglycan layers) were detected in the cell walls of lactobacilli suppressed by staphylococci BLIS, and rearrangement of ultrathin structure of their cytoplasm with the destruction of protein-ribosomal complex was noticed. CONCLUSION; Clinical S. aureus strains that produce BLIS, suppress antagonistic activity of lactobacilli were revealed, and morpho-physiological changes in lactobacilli target-cells damaged by BLIS were detected. Local damage of cell components manifested by the destruction of cytoplasm and formation of "cell shadows".

[Crowding-effect and attention].

We studied the influence of additional objects on recognition of the test visual objects. The test objects were stylized low-contrast letters having size 1.1, 2.1 or 4.3 ang. deg. The additional objects after 30 ms were followed by the test objects which were presented in the middle of the screen. The additional objects were digits 1-9 having size 1.3 ang. deg. The digits were presented at various distances from the centre of the screen. The observers' task was to identify both the test objects and the digits. Recognition of the test objects deteriorated when the digits were at small distances to the tests (crowding-effect). Recognition of digits deteriorated with the increasing distances from the centre of the screen; the effect was more pronounced when the tests were large. The contribution of laterals masking and attention into crowding-effect is discussed.

[Effect of cholerogen and protamine on tight junctions of rat enterocytes and colonocytes].

AIM: Study of tight junction state and ultrastructure changes of rat jejunum enterocytes and colon colonocytes under the effect of cholerogen and protamine. MATERIALS AND METHODS: Cholerogen (cholera toxin, Sigma-Aldrich, Germany) and protamine sulfate (Russia) were used in the study. The study was carried out in Wistar line rats. Effect of cholera toxin and protamine on rat intestine epitheliocytes was carried out by incubating intestine segments in the respective solutions. Ultrastructure changes caused by cholerogen and protamine in rat enterocytes and colonocytes were assessed based on ultrathin section analysis by transmission electron microscopy of the cells themselves and tight junctions between them compared with control. RESULTS: Effect of cholerogen on intestine mucous membrane epitheliocytes manifested in changes of cell ultrastructure, the form of which transformed as a result of increase of intercellular space without the destruction of tight junctions. Disappearance of cell plasma membrane lateral area folding and decrease of number of microvilli was noted. Enlargement of nuclei was noted only in individual cells. Effect of protamine on epithelial cell layer ultrastructure differed significantly from the effect of cholerogen. Increase of cell plasma membrane lateral area folding and significant enlargement of nuclei that moved to the central part of cells reaching its apical end were characteristic effects for protamine. Surface of a part of epitheliocytes lost microvilli with simultaneous destruction of tight junction structure. Protamine induced increase of folding only in colon without affecting jejunum. At the same time both of these substances caused increase of intercellular space in jejenum and colon epithelium. CONCLUSION: Differences in ultrathin structure of rat small intestine and colon epitheliocyte tight junctions under the effect of cholerogen and protamine were revealed.