Inflammation Mediators Regulate the Microbiota Resistance to Adverse Factors.

We studied the effects of IL-1ß, IL-8, TNFα, and prostaglandin E2α in concentrations typically observed in health and during inflammation on the growth of vaginal microbiota and its resistance to factors inhibiting the synthesis of proteins, nucleic acids, and peptidoglycans. An increase in the cytokine levels, characteristic of inflammation, inhibits the growth of Lactobacillus population and improves its resistance to adverse factors. The growth of the population of opportunistic microorganisms (S. aureus, E. coli) is stimulated under these conditions, while their resistance to adverse factors decreases. Hence, it seems that the cytokines regulate the behavior of the host cells and of its bacterial symbionts.

Proinflammatory Cytokines as Regulators of Vaginal Microbiota.

It was shown that IL-1ß, IL-8, and IL-6 in concentrations similar to those in the vagina of healthy women stimulated the growth of normal microflora (Lactobacillus spp.) and suppressed the growth and biofilm production by S. aureus and E. coli. On the contrary, these cytokines in higher concentrations typical of vaginal dysbiosis suppressed normal microflora and stimulated the growth of opportunistic microorganisms. TGF-ß1 in both doses produced a stimulating effects on study vaginal microsymbionts. It is hypothesized that pro-inflammatory cytokines serve as the molecules of interspecies communication coordinating the interactions of all components of the vaginal symbiotic system.

[EFFECT OF MICROSYMBIONTS ON THE ABILITY OF VAGINAL EPITHELIOCYTES TO MODIFY BIOLOGICAL PROPERTIES OF BACTERIA].

AIM: Study regularities of effects of various types of vaginal microsymbionts on the ability of mucosal epitheliocytes of the vagina to modify biological properties of bacteria. MATERIALS AND METHODS: Effect of thermo-inactivated cells of Staphylococcus aureus, Escherichia coli, H2O2-producing and H2O2-non-producing lactobacilli on the ability of primary vaginal epitheliocytes to alter growth and antagonistic activity of Staphylococcus aureus, Escherichia colt, H2O2-producing and H2O2-nonproducing lactobacilli was studied using a multi-component module system. RESULTS: Alterations of composition of vaginal epitlieliocyte exometabolites under the effect of S. aureus and E. coli was established to result in a pronounced stimulation of antagonistic activity of H2O2-producing and an increase of the number of H2O2-nonproducing lactobacilli. Thermo-inactivated cells of lactobacilli stimulated production of metabolites by epitheliocytes, that suppress the growth and antagonistic activity of allochthonous microflora and stimulate similar parameters of lactobacilli. The strongest effects on the ability of vaginal epitheliocytes to alter biological properties of bacteria were rendered by H2O2-producing lactobacilli. CONCLUSION: The regularities identified allow to examine epitheliocytes and normoflora of vagina as a symbiotic system, the coordinated interaction of its components is directed on maintaining microecological stability of female reproductive tract.

[The role of pro- and antioxidants of microorganisms in regulation of symbiosis homeostasis mechanisms (on the model of human vaginal biotope)].

AIM: Study the production of bacterial pro- and antioxidants in vaginal biotope and analysis of their role in regulation of symbiosis homeostasis mechanisms. MATERIALS AND METHODS: Hydrogen peroxide, catalase inhibitors and antioxidant production in bacteria isolated from 63 women with vaginal eubiosis and 53--with dysbiosis were studied. Production of pro- and antioxidants was regulated by lactate, volatile fatty acids, polyamines and Lactobacillus plantarum and Corynebacterium minutissimum polysaccharides, metabolite bactericidity of peroxide producing lactobacilli was enhanced by addition of iron (II) ions. RESULT: A high level of pro- and antioxidant production was noted for eubiosis state, and their ratio was close to 1, for biotopes with dysbiosis a multiple predominance of microbial antioxidant levels over pro-oxidants was characteristic. Peroxide producing symbionts are an important component of system of generation of hydroxyl radicals that are highly effective wide specter disinfectants. CONCLUSION: Maintenance of balance of pro- and antioxidant production by symbionts determined vaginal biotope symbiosis homeostasis. The detected high biocidic activity of hydroxyl radicals that are formed from hydrogen peroxide of normoflora through the creation of optimal conditions for their generation by selection of the respective concentrations of iron (II) ions and H2O2 opens perspective of development of novel disinfection methods.

[Effect of metabolites of H2O2-producing lactobacilli on functional activity of lysozyme].

AIM: Study the effect of metabolites of H2O2-producing lactobacilli on enzymatic and bactericidal activity of lysozyme. MATERIALS AND METHODS: 9 H2O2-producing vaginal lactobacilli, Micrococcus luteus NCTC 2665, Escherichia coli State Institute of Standardization and Control No 240367, Lactobacillus acidophilus Institute of Cellular and Intracellular Symbiosis No 37 were used. Ability of lactobacilli to produce H2O2 was evaluated by oxidation of tetramethylbenzidine by peroxidase. Lysozyme was modified by mixing with equal volumes of lactobacilli metabolites, metabolites of H2O2-producing vaginal lactobacilli previously treated with catalase were used in control. Lysozyme enzymatic activity was determined by speed of M. luteus lysis, bactericidal--by survivability of E. coli in Endo medium and L. acidophilus--in MRS medium. RESULTS: Decrease of enzymatic activity of lysozyme due to its contact with H2O2-producing lactobacilli metabolites was detected. This effect is accompanied by growth of bactericidal activity of lysozyme against E. coli and decrease against L. acidophilus. The degree of changes of enzymatic and bactericidal activity of lysozyme by lactobacilli metabolites depended on concentration of hydrogen peroxide in them. CONCLUSION: Modification of lysozyme by H2O2-producing lactobacilli metabolites resulting in opposite changes of its activity against autochthonous and allchthonous bacteria is one of the mechanisms of formation of stable microbial biocenosis in human organism.

[Role of dominant microflora in mechanisms of woman vaginal biotope protection].

AIM: Detection of properties of the dominant microsymbiont emerging in the course of interaction of associative symbiosis components and ensuring protection of vaginal mucosa from seeding by pathogens. MATERIALS AND METHODS: H202-producing Lactobacillus spp., Corynebacterium spp., Staphylococcus aureus, Escherichia coli and primary epitheliocytes from lower reproductive tract of women were used. The ability of Lactobacillus spp. to influence changes of synthesis of catalase inhibitors by corynebacteria, bactericidal activity of lysozyme, lactoferrin and thrombocyte antimicrobial protein, antimicrobial activity of primary vaginal epitheliocytes was evaluated. Changes of antagonistic and growth properties oflactobacilli during interaction with secretory products of Corynebacterium spp., S. aureus, E. coli and vaginal epitheliocytes were also studied. RESULTS: Exometabolites of both epitheliocytes and corynebacteria were revealed to stimulate in most cases lactobacilli biomass growth and Sincreased their antagonistic activity against S. aureus u E. coli. Metabolites of H202-producing lactobacilli increased synthesis of antimicrobial substances by epitheliocytes and potentiated bactericidity of natural resistance factors. Under the influence of lactobacilli metabolites an increase of production of catalase inhibitors by corynebacteria was revealed. CONCLUSION: Symbiotic interrelations of dominant microorganisms with host organism and associants under the condition of associative symbiosis leading to stimulation of production and potentiating of the effect of antibacterial protection factors are the basis for colonization resistance of vaginal biotope.

[Influence of corynebacteria metabolites on antagonistic activity of H2O2 producing lactobacilli].

AIM: Study combined influence of Corynebacterium genus bacteria metabolites and H2O2 producing lactobacilli on survival rate of Staphylococcus aureus, Escherichia coli and Lactobacillus acidophilus. MATERIALS AND METHODS: The ability to inhibit catalase of the test strains used and to reduce bactericidal effect of hydroxyl radical were determined in corynebacteria. H2O2 containing metabolites were obtained by cultivating lactobacilli in mineral medium, the amount of H2O2 was determined by oxidation of TMB by peroxidase. Bactericidal effect of lactobacilli metabolites for test strains treated by corynebacteria metabolites was evaluated by seeding results. Results. Inhibitio by corynebacteria metabolites of S. aureus catalase activity by 30-40% and E. coli catalase activ ity by 40-70% was shown. A reduction of bactericidal effect of hydroxyl radicals by corynebacteria metabolites by 30-35% for S. aureus, 38-42% for E. coli and 70-73% for L. acidophilus was noted. The enchantment of bactericidal effect of lactobacilli after treatment of the test strain by corynebacteria metabolites against S. aureus and E. coli manifested by reduction of the numbe of viable cells by 2-3 lg CFU. For L. acidophilus the bactericidal effect oflactobacilli metabolite in the same conditions reduced, and that led to the increase ofviability by 2-4 lg PFU. CONCLUSION: A conclusion on the possibility of regulation by associative bacteria the manifestations of antagonistic activity of H2O2 producing dominant microorganisms is made based on the data obtained.

[Influence of active forms of oxygen on adhesive characteristics and biofilm production by bacteria].

AIM: Study of influence of low concentrations of active forms of oxygen on the ability of bacteria to form biofilms and their adhesive characteristics. MATERIALS AND METHODS: Modification of the studied properties of bacteria was performed by treatment of the bacteria with hydrogen peroxide or hydroxyl radicals at concentrations of 0.5 mM that causes death of 5 - 7% of cells and non-lethal - 0.05 and 0.005 mM. Adhesive characteristics of Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Klebsiella pneumoniae bacteria were determined by using human erythrocytes. The degree of biofilm formation in plates modified by collagen from human fibroblasts was determined by the amount of bound crystal violet. RESULTS: A significant decrease (7 - 17%) of the adhesion of E. coli, S. aureus, K. pneumoniae to erythrocytes under the influence of hydrogen peroxide and hydroxyl radicals at the concentrations of 0.5 and 0.05 mM when compared with the control was detected. 11 - 40% stimulation of E. coli, S. aureus, K. pneumoniae biofilm formation by active forms of oxygen in all the used concentrations with simultaneous insignificant biomass growth increase was noted. Active forms of oxygen did not influence significantly the studied properties of B. subtilis. CONCLUSION: Active oxygen forms have regulatory influence on the character of interaction of bacteria with surfaces colonized by them, that determines structural-functional state of microbial biocenoses.

[Role of associative microsymbionts in functioning of associative symbiosis].

AIM: Study the role of associative microsymbionts in biocenosis based on comparative evaluation of interbacterial and epithelial-bacterial interactions on the example of vaginal biotope. MATERIALS AND METHODS: Corynebacterium spp., Staphylococcus aureus, Escherichia coli and Lactobacillus spp. and primary epitheliocytes isolated from the lower part of reproductive tract of women were used. Interactions by associant-macropartner vector were evaluated by changes of growth properties and antagonistic activity of the associant under the influence of exometabolites of vaginal epitheliocytes and expression of cytokines by epitheliocytes under the influence of associants. The nature of interaction by associant-dominant vector was evaluated by changes of adhesive properties and biofilm formation of associants and bactericidal activity of peroxide-producing lactobacilli. RESULTS: Groups of associants that are opposite by their function in symbiosis were isolated. An example of associants that stabilize associative symbiosis by stimulating growth and antagonistic activity of dominant, moderate increase of cytokine production by epitheliocytes are corynebacteria, whose growth, biofilm formation, adhesion and antagonism during interaction with dominant and macropartner are also increased. The nature of interaction of associants of the other group (S. aureus and E. coli) with dominant and macropartner is the opposite. The determinative role of hydrogen peroxide in mechanisms of differentiation of associative microsymbionts and functioning of associative symbiosis ofvagina was shown. CONCLUSION: The proposed approach allows to differentiate associative microsymbionts with various ecological roles, evaluate their contribution into maintenance of the stability of the symbiosis and open new possibilities of management of quality of microbiocenosis.

[Mechanisms of protective action of bacterial intracellular metabolites for lowering of bactericidal effect of hydroxyl radicals].

AIM: To study mechanisms of attenuation of bactericidal effect of hydroxyl radicals by bacterial intracellular metabolites. MATERIALS AND METHODS: Culture liquids of 16 strains of Lactobacillus spp., 21 strains of Corynebacterium spp., 8 strains of Micrococcus spp. and 17 strains of Staphylococcus spp. and fractions obtained from them were used. Bactericidal effect of hydroxyl radicals on Escherichia coli was measured by survival of the bacteria. RESULTS: High prevalence of ability of bacteria to prevent other microorganisms from bactericidal action of hydroxyl radicals produced in Fenton's reaction was revealed. This effect was accompanied by other two: ability to oxidize Fe2+ in Fe3+ and production of substances with antioxidant properties - extracellular polysaccharides, pigments and lipid-containing compounds. CONCLUSION: It was assumed that defense of microorganisms composing microbiocenosis from toxic effect of hydroxyl radicals realizes through production of bacterial metabolites which inactivate hydroxyl radicals or prevent their formation.

[Characteristics of microecological disturbances during endometritis].

AIM: To study the state of vaginal microbiocenosis in women with endometritis and to assess association between microecological disturbances and characteristic of clinical course of the disease. MATERIALS AND METHODS: Two hundred women of childbearing age with endometritis were studied, of which 83 had acute form of the disease, and 117 - chronic form. Control group was comprised by 30 clinically healthy women. Antilysozyme, anticomplement, and antilactoferrin activities were determined in isolated and identified microorganisms. Microecological disturbances in vaginal biotope were determined applying criteria proposed by Sokolova K.Ya. et al. and Amsel R. et al. RESULTS: Microecological disturbances in vaginal biotope were revealed on women with endometritis - in particular, bacterial vaginosis, deficiency of dominant microflora (lactobacilli), and the presence of associate microorganisms with clearly expressed persistence characteristics. On the background of microecological disturbances course of endometritis was characterized by high rate of relapses and reduction of remission period. CONCLUSION: Association of endometritis with different types of dysbiotic states of vaginal biotope could be used for prediction of the disease course as well as for correction of microecological disturbances during treatment of endometritis.

[Influence of active forms of oxygen on antilysozyme activity of bacteria].

AIM: To study the effect of active forms of oxygen on bacterial population structure on antilysozyme characteristic. MATERIALS AND METHODS: Antilysozyme activity of bacterial clones obtained after their treatment by hydrogen peroxide and hydroxyl radical in sublethal (LD10) and non-lethal concentrations. RESULTS: Shift of bacterial population structure in direction to increased proportion of clones with high level of antilysozyme activitywith elevating average population level of this characteristic under the influence of sublethal (LD10) concentrations of hydrogen peroxide and hydroxyl radical was established. Using of non-lethal concentrations of active forms of oxygen resulted in decrease of average population level of antilysozyme activity and emergence of clones with lower level of expression of this characteristic than in control population. CONCLUSION: It was assumed that active forms of oxygen provide variety of types of interaction of microorganisms with each other and with macroorganism due to change of intrapopulation heterogeneity of biological characteristics of bacteria, which enables them to realize their persistence potential.

[Active forms of oxygen as a factor regulating surface characteristics of bacterial cells].

AIM: Effects of sublethal concentration of active forms of oxygen (hydrogen peroxide and hydroxyl radical) on cell surface properties of Escherichia coil, Staphylococcus aureus, Bacillus subtilis, and Klebsiella pneumoniae were studied. MATERIALS AND METHODS: Hydrophobicity of cell surface measured by transition of cells from water phase to hydrocarbomic phase and by the ability of bacteria to sorb lysozyme and hemoglobin. RESULTS: Treating of bacterial cells with hydroxyl radicals compared with hydrogen peroxide resulted in more marked decrease of bacteria's hydrophobicity and reduction of their ability to sorb on the surface the lysozyme and hemoglobin. Increasing of the number of R-forms in the bacterial population after contact with hydroxyl radicals was revealed. CONCLUSION: Active forms of oxygen generated in biological systems in concentrations which are not lethal for majority of microorganisms can regulate prokaryote and eukaryote interactions by changing their surface characteristics and therefore they are an ecologic factor determining forming and existence of microbial cenoses.

Bacterial regulation of antagonistic activity of bacteria.

The phenomenon of microbial regulation of bacterial antagonism was studied using metabolites and cell walls of indicator microorganism culture as inductors. The algorithm of selection of stimulators of bacterial antagonistic activity is determined, experimental conditions are described, and methodological approach to stimulation of bacterial antagonistic activity is developed, which can be used for stimulation of probiotic antagonistic activity and for improving colonization resistance of the host organism during infection.

[Mechanisms for survival of bacteria in oxidative stress and iron ions deficiency].

The influence of culture medium Fe2+ content on the resistance of Escherichia coli to hydroxyl radicals formed in the presence of Fe2+ and hydrogen peroxide in Fenton reaction was investigated. It was founded that a lack of Fe2+ in a culture medium increased resistance of bacteria to hydroxyl radicals but not to hydrogen peroxide. The suggestion was made that the lack of Fe2+ starts up synthesis of metabolites which inactivate hydroxyl radical or block Fe2+ ions participating in Fenton reaction. The phenomenon under study is considered to be a possible mechanism for survival of bacteria in oxidative stress and iron ions deficiency.

[The effect of the intra- and extracellular metabolites of microorganisms isolated from various ecotopes on the catalase activity of Staphylococcus aureus 6538P]. / Izmenenie aktivnosti katalazy Staphylococcus aureus ATCC 6538P pod vliianiem metabolitov mikroorganizmov, vydelennykh iz raznykh ékotopov.

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.

[Effect of vaginal Lactobacillus metabolites on biological properties of Staphylococcus epidermidis and Escherichia coli]. / Izmenenie biologicheskikh svoistv Staphylococcus epidermidis i Escherichia coli pod vliianiem metabolitov vaginal'nykh laktobatsill v éksperimente.

The influence of supernatants and cell extracts of vaginal lactic acid bacilli on the growth, catalase, antilysozyme and anticomplementary activity of S. epidermidis and E. coli was studied. Changes observed in the persistence characteristics of these microorganisms were found to develop in different directions: in S. epidermidis a decrease in antilysozyme activity was mainly observed, while in E. coli the suppression of anticomplementary activity was registered. The modifying influence of lactic acid flora, revealed in this investigation, is regarded as one of the mechanisms facilitating the formation of microbiocenosis and ensuring colonization resistance.

Effects of microbial metabolites on catalase activity and growth of Staphylococcus aureus 6538 P.

The effects of cell extracts and supernatants of Lactobacillus spp. and Corynebacterium spp. on catalase activity and growth of Staphylococcus aureus 6538 P were studied. Intra- and extracellular metabolites of lactobacilli and corynebacteria inhibited catalase activity of S. aureus 6538 P. The growth of S. aureus 6538 P decreased after incubation with lactobacillus metabolites. The inhibitory effect of intra- and extracellular metabolites of lactobacilli and corynebacteria on catalase activity of S. aureus is a possible pathway of microbial interrelations responsible for the formation and/or development of microbial biocenoses.