Probiotic Potential, Safety Properties, and Antifungal Activities of Corynebacterium amycolatum ICIS 9 and Corynebacterium amycolatum ICIS 53 Strains.

The purpose of this study was to evaluate the probiotic characteristics and safety and to study the antifungal activity of C. amycolatum ICIS 9 and C. amycolatum ICIS 53 against Candida spp. The probiotic potential and safety properties were assessed by standard parameters. Both strains showed good survival at pH 3 for 3 h and high tolerance to 0.3% bile salts after 4 h of incubation. The indicators of hydrophobicity, autoaggregation, and surface tension for ICIS 9 were 89.43% (n-hexane) and 73.96% (xylene) and ranged from 13.13 to 39.86% and 34.27 mN/m, respectively. For ICIS 53, they were 59.95% (n-hexane) and 45.68% (xylene), from 35.58 to 51.53% and 32.40 mN/m, respectively. The strains ICIS 9 and ICIS 53 exhibited varying levels of coaggregation with all eight examined bacterial pathogens. The ICIS 9 strain was resistant to amikacin, amoxicillin, clarithromycin, chloramphenicol, ciprofloxacin, and gentamycin. ICIS 53 was resistant only to ciprofloxacin. The cell-free supernatant of strains ICIS 9 and ICIS 53 showed good antimicrobial and antibiofilm activity against 10 pathogenic vaginal and intestinal isolates of Candida spp. The CFS of ICIS 9 was more active against intestinal isolates, and the CFS of ICIS 53 showed good antimicrobial activity against vaginal isolates while inhibiting the growth of 2 out of 5 Candida spp. isolated from the intestine. Both of the strains were capable of reducing the biofilm formation of Candida fungi. In the case of the vaginal isolates of C. krusei V1, the results showed that the inhibition levels of ICIS 9 and ICIS 53 were 36.75 and 11.4%, respectively. In the case of C. albicans (V2, V3, V7, and V8), the inhibition of biofilm formation was no more than 7.07%. ICIS 9 and ICIS 53 also significantly inhibited biofilm formation of C. krusei 2613 intestinal isolates by 42.75 and 41.87%, respectively, with ICIS 9 inhibiting biofilm formation of C. albicans (2607, 2311, 2615, and 2615) from 3.38 to 15.69% and ICIS 53 from 5.95 to 23.48%. None of the strains showed DNase, haemolytic, or gelatinase activities. The results obtained revealed that ICIS 9 and ICIS 53 have safe properties and have the potential to be developed as probiotics.

[PHYSIOLOGICAL FEATURES OF CORYNEBACTERIA OF FEMALE REPRODUC- TIVE TRACT].

Literature data and results of authors' research on biological properties of corynebacteria of reproductive tract of women are analyzed. General characteristics of microorganisms is given. 20 species of corynebacteria are presented: C. amycolatum, C. aquaticum, C. aurimucosum, C. bovis, C. glucuronolyticum, C. coyleae, C.freneyi, C.jeikeium var. genitalium, C.jeikeium var. pseudogeni- talium, C. lipophiloflavum, C. kutscheri, C. minutissimum, C. nigricans, C. pseudodiphtheriticum, C. pseudotuberculosis, C. renale, C. striatum, C. tuberculostearicum (lipophile) (includes most CDC group G-2 strains), C. xerosis and C. urealyticum. Mechanisms and factors ensuring the ability of corynebacteria to exist in vaginal biotope regardless of microecological condition- the presence of high resistance to factors of innate immunity (lysozyme, complement, immunoglobulins), pH- dependent adhesion to fibronectin and vaginal epitheliocytes - are examined. The role of fi- bronectin in adhesion of bacteria to vaginal epithelial cells is described. Corynebacteria exome- tabolites are shown to facilitate maximal realization of antagonistic activity of vaginal peroxide-producing lactobacilliby supressing catalase ofopportunistic microorganisms-symbionts, that directly influences the quantity and structure of bacterial population by suppressing growth and biofilm-formation. The materials provided give evidence on the significant role of corynebac- teria in realization of physiological phenomenon - colonization resistance and allow us to exam- ine these microorganisms as an integral part of normal microbiota of woman reproduction tract.

[ROLE OF BIOLOGICAL PROPERTIES OF CORYNEBACTERIA IN ASSOCIATIVE SYMBIOSIS].

Microorganisms of the Corynebacterium genus are examined in the review as a component of a single microecological system of humans in the context of their interaction with the macroorganism, dominant and associative microorganisms under the conditions of both normo- and pathocenosis. Adhesive ability, antagonistic activity, pathogenicity and persistence factors, antibiotics resistance are described. The role of non-pathogenic corynebacteria in the formation of microbiocenoses of human body and realization of colonization resistance is shown on an example of vaginal biotope.

[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.

[The role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes].

AIM: Determination of the role of fibronectin in adhesion of corynebacteria to vaginal epitheliocytes. MATERIALS AND METHODS: Corynebacterium genus microorganisms and primary epitheliocytes isolated from the lower reproductive tract of women were used. Adhesive ability of corynebacteria was studied in polystyrene plates against fixed fibronectin and on the model of vaginal epitheliocytes. Changes in adhesion of corynebacteria to vaginal epitheliocytes was evaluated after treatment of the latter with fibronectin. RESULTS: All the studied strains had the adhesion ability to fibronectin and vaginal epitheliocytes. The same strains were attributed to groups of high, moderate or low adhesive using both plate method and method utilizing vaginal epitheliocytes model, that tells of their comparability. During the addition of fibronectin to epitheliocytes, an enhancement of adhesion of all the studied corynebacteria strains took place. Adhesion index in strains isolated from healthy women increased by an average of 46.6%, adhesion index by 10.5 bact. cells/epith. In strains isolated from women with micro-ecologic disruption, adhesion increase was by 15.3% and 4.9 bact. cells/epith., respectively. CONCLUSION: Fibronectin is a factor that determines adhesion of corynebacteria to vaginal epitheliocytes and thus is important for formation of associative symbiosis of reproductive tract of women. The data obtained open perspective of use of fibronectin with the aim of colonizing ability increase of probiotics.

[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.

[Production of cytokines by vaginal epitheliocytes in the process of interaction with dominant and associative microsymbionts].

AIM: Study the expression of cytokines by vaginal epitheliocytes in the process of interaction with dominant and associative microsymbionts. MATERIALS AND METHODS: IL-8, IL-6, IL-1beta and TNFalpha expression in response to interaction with heat inactivated Lactobacillus spp., Staphylococcus aureus, Escherichia coli, Corynebacterium spp. or their secretory products in comparison with basal expression of cytokines by vaginal epitheliocytes was studied. Results. Lactobacilli secretory products were shown not to influence the expression of IL-8 and IL-1beta and moderately stimulated IL-6 and TNFalpha expression. Contact of epitheliocytes with heat inactivated lactobacilli increased secretion of IL-8, IL-6 and IL-1beta and reduced TNFalpha production. Secretory products of S. aureus and E. coli caused stimulation of IL-6, IL-1beta production and practically did not change the expression of IL-8 and TNFalpha. Contact of epitheliocytes with heat inactivated S. aureus sup pressed TNFalpha production and had no influence on IL-8, IL-6 and IL-1beta expression, contact with E. colistimulated TNFalpha and IL-1beta expression and suppressed IL-6 expression. Changes in cytokine expression during interaction of epitheliocytes with corynebacteria were largely similar to the results of interaction with lactobacilli except IL-6 production that was markedly stimulated by corynebacteria secretory products. Conclusion. In epithelial-bacterial interactions dominant and associative microorganisms have a differential effect on functional status of mucosal epitheliocytes manifesting in production of cytokines that could be the basis of mucosal immunity regulation.

[Features of epithelial-bacterial interactions in bacterial vaginosis].

AIM: Study of features of interactions of mucosal epitheliocytes of vagina with dominant and associative vaginal microsymbionts in bacterial vaginosis. MATERIALS AND METHODS: Changes in growth characteristics and ability to form biofilms of Staphylococcus aureus, Escherichia coli, Lactobacillus spp. under the influence of secretory products of vaginal epitheliocytes obtained from 36 women with bacterial vaginosis and 32 practically healthy women were studied. RESULTS: Vaginal epitheliocytes of women with bacterial vaginosis were shown to change their functional activity during contact with microorganisms and their secretory products, causing differential influence on the properties of symbiont bacteria. A suppression of growth of dominant microsymbionts--lactobacilli, mostly their peroxide producing biovars, and a weakening of suppressing effect on the growth of biomass of associative microorganisms accompanied by stimulation of their ability to form biofilms was noted. Preliminary contact of an epitheliocyte with bacterial cell or its supernatant increased the intensity of the changes of properties of bacteria under the influence of secretory products of epitheliocytes. CONCLUSION: Suppression of growth of lactobacilli (mostly their peroxide producing biovars) and stimulation of the ability to for biofilms of the associative microorganisms as a result of bacterial-epithelial interactions in bacterial vaginosis determine mechanisms of formation and stability of pathomicrobiocenosis and a reduction of colonization resistance of vaginal biotope.

[Symbiotic interactions of corynebacteria and lactobacilli in realization of oxidative mechanisms of antagonism].

AIM: Study the interaction of vaginal corynebacteria and lactobacilli in realization of oxidative mechanism of antagonistic relations of bacteria. MATERIALS AND METHODS: Effect of supernatants of corynebacteria inhibiting catalase on antagonism of peroxide producing lactobacilli to Staphylococcus aureus was studied. RESULTS: High frequency (55.5 - 72.7%) of potentiating of antagonism of lactobacilli with medium and high level of hydrogen peroxide production under the effect of supernatants of corynebacteria inhibiting catalase was established. The frequency of potentiation of antagonism of lactobacilli and corynebacteriae depended on the intensity of hydrogen peroxide production and on the ability of corynebacteria to suppress catalase of staphylococci. CONCLUSION: Potentiation of antagonism to S. aureus of peroxide producing lactobacilli and corynebacteria with catalase inhibitors gives evidence on realization of oxidative bacterial mechanism of colonization resistance in human organism.

[Epithelial-bacterial interactions as a basis of microbiocenosis formation].

AIM: Evaluate significance of epithelial-bacterial interactions for the formation and/or support of microbiocenoses. MATERIALS AND METHODS: Effect of vaginal epitheliocytes on growth properties of lactobacilli strains obtained from the same biotope from 16 women with vaginal normocenosis was studied. During intravaginal probiotic therapy in 24 women with vaginal dysbiosis probability of colonization of vagina by probiotic strain was evaluated depending on the result of effect of vaginal epitheliocytes on its growth properties. RESULTS: Exometabolites of epitheliocytes in normocenosis were shown to render stimulating effect on the growth of autostrain of dominant microflora, that probably is the basis of formation and/or support of microbiocenosis. Stimulating effect of exometabolites of vaginal epitheliocytes of patients on growth properties of probiotic strain was revealed to be the prerequisite for successful probiotic therapy. CONCLUSION: The data obtained allow to examine epithelial-bacterial interactions as a basis of formation of microbiocenoses. Evaluation of these interactions may be used for individual selection of probiotic strains.

[Influence of microbial-epithelial interactions on biological characteristics of vaginal microflora].

AIM: Evaluation of influence of microbial epithelial interactions on growth properties and antagonistic activity of dominant and associated vaginal microflora. MATERIALS AND METHODS: Growth characteristics and antagonistic activity changes were studied in associated (Staphylococcus aureus, Escherichia coli, Corynebacterium spp.) and dominant (Lactobacilus spp.) microflora during contact with vaginal epitheliocytes and influence of vaginal epitheliocyte secretory products by using the developed technique. RESULTS: Secretory products of vaginal epitheliocytes and vaginal epitheliocytes differentially influence growth factors of vaginal microbiocenosis. Suppression of S. aureus and E. coli biomass growth based predominately on extracellular secretory products of epitheliocytes was observed. Stimulation of growth properties was prevalent for lactobacilli and corynebacteria and was observed in contact interaction as well as during effect of epitheliocyte extracellular secretory products. CONCLUSION: The resulting bacterial-epithelial interaction is a differentiating feature between autochthonous and allochthonous microflora due to the creation of selective advantages for dominant microsymbionts by growth properties and antagonistic activity stimulation, and biomass growth suppression of associated symbionts.

[Antibiotic resistance of coryneform bacteria isolated from the reproductive tract of women].

The species composition, antilysozyme activity and antibiotic resistance of coryneform bacteria, isolated from the reproductive tract of women with microecological disturbances, were studied. Sixty six women without microecological disturbances and 102 female patients with microecological disturbances in the reproductive tract were examined. The study showed that Corynebacterium minutissimum, C. amycolatum, C. group JK, C. bovis and C. pseudodiphtheriticum prevailed in the healthy women. In the patients with microecological disturbances in the reproductive tract C. vitarumen, C. matruchotii, C. striatum, C. renale and C. urealyticum were detected in addition to the above species. The average antilysozyme activity of the coryneform bacteria isolated from the healthy women was 1.32 +/- 0.47. In the patients with microecological disturbances in the reproductive tract it was 1.84 +/- 0.38. The in vitro susceptibility of the coryneform bacteria to antimicrobial agents was determined. High prevalence of resistance to beta-lactams (oxacillin and penicillin), erythromycin and co-trimoxazole was detected. Thus, the species variety and the antilysozyme activity of the coryneform bacteria in the reproductive tract of the women with microecological disturbances were found to be higher. The high prevalence of resistance to oxacillin, penicillin, erythromycin and co-trimoxazole in the coryneform bacteria isolated from the patients with the microecological disturbances did not differ from that in the healthy women.

[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.

[Vaginal microbiocenosis in trichomoniasis].

Taxonomic complex of vaginal microflora and its persistence characteristics in trichomoniasis and microecological disturbances were studied. Diminution of biotope colonization resistance was characterized by decreased autochthonous microorganisms' number and increased opportunistic microflora with expressed persistence. That could be of a great pathogenetic importance in the occurrence of reproductive tract inflammatory diseases of the bacterial etiology in addition of trichomoniasis in females.

[Effect of normal microflora of female reproductive tract in colonization resistance].

The role of female reproductive tract microflora in the maintenance of biotope colonization resistance was described. The role of lactobacilli possessing antagonistic properties in the reproductive tract defense was assessed. Classification of bacterial mechanisms of colonization resistance including block of the adhesion, antagonistic action of normal microflora associated with the production of antibacterial substances and suppression of allochthonous bacteria persistence characteristics was presented. Colonization resistance was considered as a physiological phenomenon of microecological homeostasis being a result of symbiotic relations of a host organism and autochthonous microflora.