[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.

[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".

[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.

[Repression of bacterial biofilms in suppurative necrotic complications of diabetic foot syndrome by ultrasound cavitation].

AIM: Study of wound microbial landscape in suppurative necrotic complications in patients with diabetic foot syndrome (DFS), and a possibility of suppression of bacterial biofilm development in these wounds by ultrasound cavitation (USC). MATERIALS AND METHODS: 2 randomized groups of patients were included in the study: main group of 145 individuals with suppurative necrotic complications of DFS, who received ultrasound cavitation therapy, and comparison group of 86 patients who received basic therapy. In the main group Sonoca-180 (Siring, Germany) unit, that allows to combine the process of mechanical wound treatment with antibacterial action of low frequency ultrasound, was used for wound treatment. Quantitative and qualitative composition of wound microflora was studied by using bacteriological method with parallel utilization of transmission and scanning electron microscopy in the dynamic of USC treatment. RESULTS: In patients with DFS bacterial biofilms containing congestions in the polysaccharide matrix and fixed at the surface of dense structure of periostenum wound were detected. In the microbial landscape in DFS patients in 21% of cases aerobic-anaerobic microflora was detected, while associations included mostly from 2 to 5 bacteria species--members of the Peptococcus, Peptostreptococcus, Prevotella, Bacteroides genera. Bacteria of Staphylococcus, Acinetobacter, Pseudomonas, Escherichia and Klebsiella sp. genera were detected less frequently. Application of USC in the main group resulted in a decrease of microbial contamination of wounds in the short-term. CONCLUSION: USC method, when compared with classical therapy approach in DFS complications, in patients with suppurative necrotic complications allows not only to remove mechanically the necrotic tissues but also to effectively suppress the bacterial biofilms that have formed in the wounds, this promotes the acceleration of recovery process and preparation of the patients for further plastic surgery intervention.

[Sorption properties of various polysaccharide matrixes to Lactobacillus plantarum 8RA-3 bacteria].

AIM: Study of sorption properties of various spherical polysaccharide matrixes designated as Spherocell to probiotic Lactobacillus plantarum 8RA-3 bacteria. MATERIALS AND METHODS: Industrial strain of L. plantarum 8PA-3 was used. The process of immobilization of lactobacilli on 3 variants of spherical sorbents was studied. The first sorbent - neutral, composed of nonpolar cellulose matrix with ("0") charge, the second--DEAE obtained by modification of cellulose by diethylaminoethyl groups with positive ("+") charge and the third--CM (carboxymethyl) with negative ("-") charge. Cellulose matrixes were designated by us by the term Spherocell. Immobilization of bacterial cells on Spherocell was performed by addition of suspension containing 1.0 x 10(9) CFU/ml. The effect of bacterial immobilization was evaluated by CFU/ ml titration and by electron microscopy. RESULTS: The dependence on matrix charge of adsorption immobilization on sorbent granules of lactobacilli cells was shown. At certain equal parameters (granule size, surface characteristics, charge value) the positively charged matrix sorbed 3-10 times more cells than neutral and 20-25 times more than negatively charged matrix. Each 100-180 microm Spherocell DEAE particle could sorb more than 1000 viable bacterial cells. CONCLUSION: Positively charged polysaccharide matrix Spherocell DEAE obtained by modification of cellulose by diethylaminoethyl groups is promising for creation of immobilized probiotic preparations.

[Microecological and electron microscopic study of skin microbiota in patients with atopic dermatitis].

AIM: Bacteriological and electron microscopic study of skin of healthy persons and patients with atopic dermatitis (AD). MATERIALS AND METHODS: Fifteen patients (7 males and 8 females aged 17-53 years) with lichenoid form of AD treated in dermatovenerologic outpatient clinics were compared with group of virtually healthy 10 persons of the same age. Comparative study of skin and electron microscopic evaluation of ultrathin sections from affected areas of the skin were performed in all participants. Main representatives of resident and facultative groups of microbiota were counted. RESULTS: Differences in microbial spectrum and level of physiological activity of microorganisms colonizing skin surface of healthy persons and patients with AD were revealed. Unlike microbes on healhy skin, the microorganisms colonizing skin of the patients with AD were characterized by profound polymorphism of cells. Widening of population spectrum of opportunistic microorganisms belonging to genuses Staphylococcus, Corynebacterium, and Candida was accompanied by manifestation of destructive changes of the skin of patients with AD. Electron microscopic evaluation of skin of patients with AD showed that bacterial cells located predominantly in upper layers of epidermis. Major number of microorganisms colonized corneal layer and located between friable skin squama. CONCLUSION: Changes of skin microbiota with marked pathomorphologic changes of keratinocytes were observed in patients with AD that necessary to consider during basic therapy of this disease.

[Disorganization of biofilms of clinical strains of staphylococci by metabolites of lactobacilli].

AIM: To study morpho-physiologic characteristics of clinical biofilm-forming strains of Staphylococcus aureus and S. epidermidis suppressed by metabolites of lactobacilli, which produce bacteriocin-like substances. MATERIALS AND METHODS: Two clinical biofilm-forming strains of S. aureus and one strain of S. epidermidis were used. Strains Lactobacillus plantarum L3 and L. fermentum 97 were used as producers of bactericidal metabolites. Ability of staphylococci to form biofilms was studied after their growth during 18 h at 37 degrees C in beef-extract broth with subsequent registration of biofilms attached to walls and bottom of polystyrene plate and stained by 0.1% alcohol solution of crystal violet. Ultrastructural changes in target cells were visualized by electron microscopy. RESULTS: Suppressive effect of lactobacilli metabolites on formation of biofilms by staphylococci was established. Ultrasructural changes, which characterize disorganization of vital processes such as mitosis, synthesis of DNA and peptidoglycan layer, were revealed in target cells. Formation and detachment of peptidoglycan globules from cell wall surface externally were noted. CONCLUSION: Metabolites of lactobacilli are able to suppress formation of biofilms and induce ultrastructural changes in S. aureus and S. epidermidis, which lead to their destruction.

[Ultrastructural change of lactobacilli during suppression of their growth by clinical strains of Candida albicans].

AIM: Detection of morphophysiological changes in cells of manufacturing strains of lactobacilli suppressed by antagonistically active clinical cultures of Candida albicans, isolated from women with torpid course of vaginosis and vaginitis. MATERIALS AND METHODS: Three clinical strains of C. albicans suppressing growth of bacterial cells from manufacturing strains of Lactobacillus plantarum 8-PA-3, L. fermentum 90-4C and L. acidophilus D75 on modified agar-added nutrient medium were used. Using electron microscopy, ultrastructural changes in lactobacilli suppressed by C. albicans were studied. RESULTS: In places of tight contacts of interacting microorganisms, destructive changes in cell walls of lactobacilli appeared as desquamation of small layerwise fragments of peptidoglycan layers were observed. Rearrangement of ultrafine structure of lactobacilli's cytoplasm which expressed as destruction of protein-ribosomal complex was also noted. Local defects of cellular components led to total destruction of cytoplasmic contents and appearance of "cellular shadows". CONCLUSION: Surmounting by C. albicans the antagonistic action of lactobacilli with destruction of peptidoglycan layer of their cell walls could explain persistence of yeast fungi during chronic forms of vaginosis in women.

[Using of Spherocelle sorbents for construction of immobilized probiotics].

AIM: To assess sorption properties of Spherocelle beads consisting of particles of macroporous celiulose with various charges in relation to bacterial cells of manufacturing probiotic strains from different taxonomic groups. MATERIALS AND METHODS: The following manufacturing strains: Bifidobacterium bifidum 1, Lactobacillus plantarum 8PA-3 and Escherichia coli M-17, as well as 3 variants of Spherocelles' matrix: neutral, with positive and negative charges, were used. RESULTS: Spherocelle globules DEAE with a positive charge of the matrix were successively used for designing of immobilized probiotic preparations. Efficacy of sorbent is determined by sorption of > or =1000 viable cells as well as bacterial metabolites interacting in conditions of sorbent-regulated pH on each globule with diameter 100-180 microm. It provides, on the one hand, prolonged viability of probiotic bacteria in culture fluid within 6 months and, on the other hand, optimal pharmacokinetics of preparation due to gradual desorption of metabolites from sorbent globules. CONCLUSION: Sorbent Spherocell DEAE is biocompatible with cells of manufacturing strains of lactobacilli, bifidobacteria and E. coli and recommended for designing of immobilized probiotics.

[Effect of interleukin-1beta preparation on dynamics of healing of experimental wound in mice].

AIM: To investigate the effect of application of IL-1beta on dynamics of wound healing in immunosupressed mice using electron microscopy. MATERIALS AND METHODS: Forty-five outbred adult male mice were used as a biological model. Immunosuppression was induced by administration of hydrocortisone. Using biopsy punches ("Stiefel"), 2 wounds 4 cm in diameter were inflicted to each animal in area of back part of the spine with control of their depth to superficial muscular fascia. Dynamics of changes in regenerating epidermis and identification of microbial communities were studied by electron microscopy of ultrathin sections. The speed of skin regeneration was evaluated by photometric method. Photometry of the wound was performed just after wound-inflicting operation and on 3rd, 8th, and 14th day of the experiment. RESULTS: Electron microscopy showed that IL-1beta promoted cell differentiation processes that was expressed in earlier development of protein-synthesizing organelles of newly generated spinous cells and in formation of stratum granulosum with greater number of intracellular junctions. The most expressed differences in wound regeneration were observed in the samples taken on 8th and 14th days of the experiment. CONCLUSION: Application of IL-1beta promotes wound healing under immunosuppression and accelerates purification of the wound from microorganisms preventing growth of gram-positive and gram-negative microflora as well as development of filamentous forms of micromycetes.

[The electron microscopic study of cell-to-cell interactions between antagonistic microorganisms].

The electron microscopic study of thin sections and positively stained specimens of cells taken from particular cocultures of Lactobacillus acidophilus D75, Lactobacillus casei YIT 9018, Shigella flexnery 2a, Bacillus subtilis ATCC 6633, and Staphylococcus aureus ATCC 25923 (some of these bacteria are antagonistic to others) showed the presence of specific ultrastructural elements indicating cell specialization and cooperation. The responses of antagonistic bacteria manifested themselves at the cellular and population levels.

[Ultrastructural changes in Shigella flexneri cells during interaction with bacteriocinogenic Lactobacillus acidophilus].

Results of electron-microscopic examination of Shigella flexneri cells, subjected to influence the bacteriocin-producing Lactobacillus acidophilus bacteria are presented. The response of shigellae to bacteriocinogenic lactobacilli was shown both on cellular and population levels. On population level the correlation of various morphological types of shigella cells with increase of involution, lysing and resting forms is revealed. At a cellular level the specific ultrastructural changes of shigella evidencing the significant destructive processes of the cells were revealed. In one case destabilization of shigella cellular wall was observed, that was manifested in expansion of periplasmic spaces and appearence of specific involution forms of the cells. In other cases, changes in the ultrastructural organization of shigella nucleoid were found out, manifested in disappearance of thin-fibrillar DNA and formation of electronic-dense globular structures of the cells.

[Antagonistic effect of bacteriocinogenic Lactobacillus acidophilus on Klebsiella pneumoniae, Citrobacter freundii and Proteus mirabilis cells].

Data of the ultrastructural cellular changes of conditionally pathogenic enterobacteria, including K. pneumoniae, C. freundii and P. mirabilis cells impacts to bacteriocin-producing L. acidophilus are presented. Enterobacteria in response to the bacteriocinogenic effect of lactobacilli are manifestated by expressive destructions of sensitive to pore formation bacteriocin cells. Various morphological types of enterobacteria cells with increase of involution, lysing and resting forms are revealed. The specific ultrastructural changes of enterobacteria cells which evidencing the significant destructive processes of the cells membranes are detected. The destabilization of cellular wall in expansion periplasmic spaces and appearance of the ultrastructural reorganization of bacterial cells nucleoid also are registrated. Revealing the mechanism of lactobacilli secreted bacteriocin action to conditionally pathogenic enterobacteria might provide new ways to select the effective highly antagonistic probiotic strains.

[The effect of isoproterenol on protein extrusion from tissue explants of the mouse mamma]. / Vliianie izoproterenola na ékstruziiu belka iz éksplanta tkani molochnoi zhelezy myshi.

Culture of mouse mammary gland cells was treated with beta-agonist isoproterenol to result in dose dependent increase in protein. Propranolol, beta-antagonist, blocked this effect of isoproterenol. According to electron microscopic analysis, application of isoproterenol leads to disappearance of vesicles from apical zones of the secretory cells, and to increase in casein micelles in alveolar lumen. As follows from the obtained data, catecholamines play part in regulation of mammary gland activity, stimulating process (via beta-adrenoreceptors) of protein extraction from secretory cells of the mouse mammary gland.

[Structure of an Escherichia coli bacterial suspension]. / Struktura bakterial'noi suspenzii Escherichia coli.

The structure of bacterial suspensions of Escherichia coli M-17 at the counting concentrations of the cells 10(7), 10(8), 10(9) i/ml and in the temperature range of (18-50) degrees C has been investigated by means of orientational conductometric, electron microscopic and UV-spectroscopic methods. On the basis of experimental relationships of the anisotropy of suspensions electric conductivity upon the intensity of a sinusoidal electric field and relaxation of anisotropy after switching off the field the function of the distribution of bacteria with respect to their sizes was evaluated at different temperatures and concentrations. The conductometric function of bacteria distribution is in a good agreement with the analogous function obtained with the help of the electron microscope. In accordance with the functions the suspension of E. coli contained three kinds of cells: high electronic density, low electronic density bacteria and bacteria aggregates. Relative amounts of every kind of bacteria depended on temperature and concentration of cells. The minimum of bacteria aggregates and maximum of low electronic density cells were obtained in the temperature range of (32-42) degrees C. This fact could be explained by the activation of the transport membrane systems in this temperature range. This hypothesis was confirmed by the UV-spectroscopic method.

[Temperature changes of extracellular media state on a bacterial suspension]. / Temperaturnye izmeneniia sostoianiia vnekletochnoi sredy v bakterial'noi suspenzii.

Within temperature intervals 30-40 degrees C for bacterial suspension of E. coli and 24-34 degrees C for B. flavum the extracellular medium exists in a specific state. Water in the extracellular medium is stabilized by increased hydrophobicity of extracellular protein molecules surface due to proteins conformational change. The total amount of UV-absorbing metabolites is decreased as a result of activation of microorganisms transport systems. The temperature intervals of these processes are different for both types of the microorganisms and coincide with their temperature optima of vital activity.

[Contacts between the cells in bacterial colonies]. / Kontakty mezhdu kletkami v bakterial'nykh koloniiakh.

The interaction of cells in microbial colonies has been studied by electron-microscopic techniques. Two types of contacts between cells have been found to exist in the colonies of Gram-negative bacteria of the genera Escherichia, Shigella and Salmonella: close cell adhesion due to the fusion of cell-wall outer membranes and the formation of intersections consisting of membranous tubules. At the sites of close adhesion the fusion of cytoplasmic and outer membranes have been found to occur in Bayer's zones. In the colonies of Gram-positive bacteria of the genera Staphylococcus and Brevibacterium only one type of contacts has been revealed: the fusion of the peptidoglycan layers of the cell walls. The results of this study indicate that in colonies bacteria are not completely isolated; their interaction leads to the formation of a three-dimensional structure denoted as a cooperative cell system.

[Effect of polyamines on the thermal resistance of Escherichia coli cells in heat shock]. / Vliianie poliaminov na termoustoichivost' kletok Escherichia coli pri teplovom shoke.

Polyamines intensify the effect of a heat shock on Escherichia coli M-17 cells. The lethal effect of polyamines rises in the spermidine--spermine series as their concentration and the duration of heat action is increased. The effect of polyamines on the cells subjected to a heat shock is not associated with the activity of amine oxidases and, apparently, does not destabilize the membranes. As was demonstrated using electron microscopy, the cells undergo morphological changes in the presence of polyamines in the course of a heat shock; in particular, electron-dense regions appear in the nucleoid zone, presumably, due to DNA conformational rearrangements during the shock in the presence of polyamines.

[Significance of rec A-gene activity in altering the ultrastructure of Escherichia coli]. / Znachenie aktivnosti rec A-gena v izmenenii ul'trastruktury Escherichia coli.

The pathogenic E. coli strain, serovar 0124 studied in this investigation and its mutants rec A 56 and rec A 441 differ in their ultrastructural organization. In strains having defects in gene rec A, the appearance of intracytoplasmic membranous structures and a great number of extracellular membranous vesicles, as well as the formation of the filamentous forms of cells in the colonies, can be observed. These data indicate that the product of gene rec A plays an active role in the metabolism of bacterial membranes.