Isolation and structural identification of glycopolymers of Bifidobacterium bifidum BIM B-733D as putative players in pathogenesis of autoimmune thyroid diseases.

Bifidobacterium bifidum 791 (commercially available as B. bifidum BIM B-733D) cell-surface biopolymers (BPs) interact selectively with human serum thyroid peroxidase (TPO) and thyroglobulin (Tg) autoantibodies (anti TPO and anti Tg, respectively). BPanti-TPO and BPanti-Tg were isolated from the soluble fraction of B. bifidum BIM B-733D by affinity chromatography with anti-TPO or anti-Tg, respectively. Homogeneity of affinity eluates (AEanti-TPO and AEanti-Tg) was tested by size exclusion chromatography. For each AE, the elution profiles generated on the basis of absorbance at 280 nm do not conform to ELISA data for functional activity characteristic of BPs. Moreover, high functional activity was detected in chromatographic fractions that had significantly different molecular weights and no absorbance at 280 nm, which suggests a non-protein (carbohydrate) nature of BPanti-TPO and BPanti-Tg. The semi-preparative size exclusion chromatography of AEanti-TPO and AEanti-Tg with detection by refractometer gave 5,000-7,000 Da fractions containing substances that interact selectively with either anti TPO (BPanti-TPO) or anti-Tg (BPanti-Tg) according to ELISA data. Analysis by two-dimensional NMR spectroscopy including a 1H, 13C-heteronuclear single-quantum coherence experiment indicated that both substances are linear α-1,6-glucans. For the first time, an immunological similarity (molecular mimicry) of glycopolymers of B. bifidum BIM B-733D and human thyroid proteins, TPO and Tg, was shown. On the whole, our data point to a possible role of bifidobacteria in the pathogenesis of autoimmune thyroid diseases (ATD). The main requirements for triggering/acceleration or prevention/abrogation of ATD by bifidobacteria through molecular mimicry mechanism are hypothesised to be (1) genetic predisposition to ATD and (2) intestinal epithelium penetration by α-1,6-glucan.

The role of components of Bifidobacterium and Lactobacillus in pathogenesis and serologic diagnosis of autoimmune thyroid diseases.

During recent years, researchers have been focusing on the concept of an infectious etiology of autoimmune diseases. The most discussed theory is molecular mimicry, i.e. the emergence of autoreactive clones of T- and B-lymphocytes as a result of cross-immune response to homologous bacterial or viral antigen. Information on the role of probiotic microorganisms (PM) in the molecular mechanisms of autoimmune thyroid diseases (ATD) is limited. Using proteins and immunogenic peptides databanks and relevant computer programs, the homology between the amino acid sequences of thyroid peroxidase (TPO) and thyroglobulin (Tg), which are potential B- and T-cell epitopes of these antigens, and proteins of bifidobacteria and lactobacilli was established. Moreover, we have found components of cells of Bifidobacterium bifidum 791, Bifidobacterium adolescentis 94 BIM, Bifidobacterium longum B379M and Lactobacillus plantarum B-01 that selectively bind human antibodies to TPO (anti-TPO) and antibodies to Tg (anti-Tg) and compete with natural antigens for the binding of anti-TPO and anti-Tg in ELISA. Additionally, a three-fold difference was observed between the probability of detecting antibodies (Abs) to the antigens of L. plantarum B-01 and B. bifidum 791 in serum samples containing and those not containing anti-TPO. On the whole, our data are arguments in favour of the assumption of the possible role of PM of the genera Bifidobacterium and Lactobacillus in triggering ATD by the mechanism of molecular mimicry. The data obtained in silico and in vitro should be proven by use of animal models and clinical studies for extrapolations to the whole body. Possible antigenic properties of components/proteins of bifidobacteria and lactobacilli, selectively binding anti-TPO and anti-Tg should be taken into consideration. Natural human Abs to these bacterial components are probably able to cross-react with the TPO and Tg in the ELISA for detection of anti-TPO and anti-Tg, which are serologic markers of ATD. It can lead to unspecific false positive results and, hence, to an incorrect diagnosis.

Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell.

Bacteriophages are an attractive tool for application in the therapy of bacterial infections, for biological control of bacterial contamination of foodstuffs in the alimentary industry, in plant protection, for control of water-borne pathogens, and control of environmental microflora. This review is mainly focused on structures governing phage recognition of host cell and mechanisms of phage adsorption and penetration into microbial cell.

[Application of the methods of molecular systematics to classification and identification of bacteria of the genus Bifidobacterium].

The methods of molecular systematics currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach to taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research into molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.

[Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance].

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular beta-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60-70% of beta-galactosidase and alpha-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30-90%.

[Fractions of barley spent grain as media for growth of probiotic bacteria].

The application of the protein and polysaccharide fractions of barley spent grain as a basis of growth media for probiotic bacteria was studied. High values of biomass yield, cell viability, and organic acid production were observed in the variants of media containing the barley spent grain supplemented with lactose, ascorbic acid, yeast extract, and mineral salts. Cells of lactic acid bacteria and bifidobacteria had the typical rod-shaped morphology.

[Biological activity of probiotic microorganisms].

Adaptation of bifidobacteria and lactic acid bacteria to nutrient media with increased concentrations of bile (1%) and protein substrates of animal origin allowed the variants resistant to bile and displaying a high production of proteolytic enzymes (active within the pH range of 2.5-9.0) to be selected. Administration of the preparations involving the selected bifidobacteria and lactic acid bacteria assisted in the normalization of the intestinal microflora and activation of protein metabolism in the organism of animals. Specifically, it increased the total protein level in blood serum and redistributed protein fractions, increasing the content of globulins and decreasing albumin concentration.

[Analysis of phospholipids in bifidobacteria].

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modem procedures for identifying Bifidobacterium species.

[Isolation and comparative analysis of glycolipid fractions in bifidobacteria].

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (Rf) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875T), Rhodococcus equi PCMT 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H2O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H2O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G1 and G2), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.

[Biological activity of polar lipids from bifidobacteria].

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.

[Characterization of the cell-bound polysaccharides of Bifidobacterium adolescentis 94 BIM]. / Kharakteristika polisakharidov, sekretiruemykh Bifidobacterium adolescentis 94 BIM.

The cell-bound polysaccharides (PSs) of Bifidobacterium adolescentis 94 BIM were stripped from the cell surface with 2% sodium dodecyl sulfate (SDS), 1.5% Cetavlon, and 1% Triton X-100 and purified by precipitation with 5 volumes of ethanol. According to the extraction conditions used, the polysaccharide preparations were designated as PS-SDS-6 degrees C, PS-SDS-100 degrees C, PS-Cet, and PS-Trit. The gel-permeation chromatography of the first two preparations with the use of a Bio-Gel P-10 column and 1% acetic acid as the eluant yielded two peaks, F1 and F2, which contained carbohydrates and no phosphorus. All polysaccharides were primarily composed of glucose and galactose. The polysaccharides PS-Cet and PS-Trit were found to be branched and contain glucose residues at the terminal position, position 4, and position 6, and galactose residue at position 3. PS-SDS-6 degrees C has a glucose residue at position 4.

[Study of the physiological and biochemical characteristics of bifidobacteria at the late stages of population development]. / Issledovanie fisiologo-biokhimicheskikh osobennostei bifidobakterii na pozdnikh stadiiakh razvitiia populiatsii.

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidum no. 1, B. adolescentis MC-42, and B. adolescentis 94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidum no. 1 in a batch mode lasted 100 h at a population density of 10(6) CFU/ml and the growth of B. adolescentis MC-42 and 94-BIM lasted 96-120 h at population densities from 10(4) to 10(7) CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with lysed cytoplasm in the cell center and intact cytoplasm in the apical parts of the cells. The maximum production of extra-cellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.

[Microelements preparations obtained during processing of natural flint affect the physiology and biochemistry of bifidobacteria]. / Vliianie preparatov mikroélementov, poluchennykh pri obrabotke prirodnogo minerala kremnia, na fiziologo-biokhimicheskie osobennosti biofidobakterii.

Microelement preparations obtained in the course of processing of flint powder stimulate the biological activity of Bifidobacterium adolescentis 94 BIM, grown on complex and synthetic nutritive media. The composition of the microelement preparations differed in the content of cations and anions. Introduction of the preparations into the cultures of physiologically active or anabiotic forms of bifidobacteria changed the parameters of exponential growth: compared to controls, the cultures were characterized by increased specific growth rate and decreased generation time. In the presence of microelements, the development of populations of bifidobacteria was associated with more pronounced accumulation of metabolic products (acetate, lactate, and ethanol). Introduction of microelement preparations increased the rate of synthesis of the extracellular proteinase (maximum content of the enzyme was observed after 3 h, whereas control cultures attained this level only after 6 h).

[Production of the therapeutic and prophylactic preparation enterobifidin on the basis of Bifidobacterium adolescentis MC-42]. / Poluchenie lechebno-profilakticheskogo preparata énterobifidin na osnove Bifidobacterium adolescentis MC-42.

Production of Enterobifidin comprises preparation of culture media, reparation of lyophilized Bifidobacterium adolescentis MS-42 culture, preparation of starters, cultivation of bacteria in fermenters, biomass conservation, and its biological control. The preparation contains physiologically active bifidobacterium cells with high activities of growth (mu = 0.7 h-1, g = 1.0 h) and acid formation (titratable acidity is approximately 120-140 degrees T; acetate concentration, 0.50-0.75%; and lactate concentration, 0.33-0.50%). The antagonistic activity of these bacteria towards Escherichia coli 08, E. coli 086, E. coli 015, E. coli 0115, and E. coli 0101 amounts to 98.2;, to Proteus vulgaris 102, to 87.2; and Staphylococcus aureus 209p, to 83.2%. The bifidobacteria (with a titer of 10(9) CFU/ml) remained viable for two to five months.

[Production of proteinases associated with Bifidobacterium adolescentis 94-BIM cell wall]. / Produktsiia assotsiirovannykh s kletochnoi stenkoi proteinaz Bifidobacterium adolescentis 94-BIM.

Bifidobacterium adolescentis 94-BIM was found to produce cell-wall bound proteolytic enzymes active at acidic, neutral, and alkaline pH values. The solubilization of proteinases with 0.5% Triton X-100 substantially improved the yield of the enzymes. The most active accumulation of cell-bound proteinases was observed in the third hour of cultivation at rates of 156.7, 179.5, and 111.1 U/(mg h), measured at pH 2.5, 7.0, and 9.0, respectively. It is suggested that the cell-wall bound proteinases of B. adolescentis 94-BIM are the precursors of the enzymes secreted into the medium.

[Structure-functional organization of bifidobacteria]. / Issledovanie strukturno-funktsional'noi organizatsii bifidobakterii.

Regularities in the development of bifidobacterial populations were studied at cellular and subcellular levels. The population development of Bifidobacterium adolescentis MC-42 and B. bifidum no. 1 was shown to be accompanied by morphological cell differentiation. A scheme of morphological transformations of bifidobacterial cells in the developmental cycle of populations is proposed. Multiplication occurs via reversion of transitory rod-shaped and coccoid forms into repeatedly budding and dichotomously branching multiseptate filaments, which, under certain conditions, fragment with the formation of differentiated reproductive forms. The morphological differentiation of bifidobacterial cells in the developmental cycle of populations correlates with changes in the cell ultrastructure. Physiologically active proliferating cells contain an intracyto-plasmic mesosomal complex, chains of polyribosomes connected with membrane structures, and abundant polyphosphate and polyglucoside inclusions. Bifidobacterial populations are highly organized mycelium-type structures, whose integrity is provided for by the cohesion of the cells. Intercellular contacts are accomplished via cell wall fusion, capsules, and cordlike and globular structures.

[Isolation and characteristics of a protein-polysaccharide complex, secreted by Bifidobacterium adolescentis]. / Vydelenie i kharakteristika belkovo-polisakharidnogo kompleksa, sekretiruemogo Bifidobacterium adolescentis.

Extracellular fibrillar protein-polysaccharide complex (PPC) of Bifidobacterium adolescentis was found to be bound to the outer surface of the cell wall. However, at the late stages of B. adolescentis cultivation (48-72 h of growth), it also occurred in the culture liquid, which can be explained by the expansion of the outer material of the cell wall. PPCs isolated from the cell surface and culture liquid contained proteins and carbohydrates in a ratio of 1:1 and from 1:4 to 1:5, respectively. PPC exerted a concentration-dependent bifidogenic effect: it stimulated growth, acidogenesis, accumulation of extracellular proteins and enzymes, and sugar utilization in B. adolescentis cells grown in synthetic Eagle's medium. PPC also promoted the rehabilitation of anabiotic forms of B. adolescentis. The PPC variants III-DN-48 and IV-DN-24, isolated from the cell surface of B. adolescentis, had the most pronounced bifidogenic effect.

[Features of growth and formation of extracellular proteinases of Bifidobacterium adolescentis 94-BIM]. / Osobennosti rosta i obrazovaniia vnekletochnykh proteinaz Bifidobacterium adolescentis 94-BIM.

Growth characteristics of a new bifidobacterial strain, Bifidobacterium adolescentis 94-BIM, were determined during cultivation in various nutrient media. The strain was obtained through selection by the criterion of proteolytic activity and was shown to synthesize acidic, neutral, and alkaline extracellular proteinases in all of the growth media tested. The growth yield of the strain varied from 2.9 x 10(8) to 1.7 x 10(10) CFU/ml with acidogenesis of up to 131 degrees T. The most rapid growth and accumulation of extracellular proteinases were observed during the first 24 h of cultivation. The maximum growth rate (0.144 h-1) was observed in the ninth hour of cultivation, whereas the most rapid accumulation of acidic, neutral, and alkaline proteinases (at rates of 53, 88.8, and 59.2 units/(mg h), respectively) occurred in the sixth hour of cultivation.

[Biological activity of the protein-polysaccharide complex secreted by Bifidobacterium adolescentis]. / Issledovanie biologicheskoi aktivnosti belkovo-polisakharidnogo kompleksa, sekretiruemogo Bifidobacterium adolescentis.

Extracellular protein-polysaccharide complex (PPC) of Bifidobacterium adolescentis stimulated growth, acidogenesis, accumulation of extracellular proteins and enzymes and the utilization of sugars in both physiologically active and anabiotic forms of B. adolescentis cultivated in rich nutrient media. The PPC variants III-DN-48 and IV-DN-24 isolated from the cell surface of B. adolescentis had the most pronounced bifidogenic effect. PPC promoted a quick and complete rehabitation of anabiotic bifidobacteria. The PPC variants III-DN-48 and IV-DN-24 were shown to be nontoxic for the population of Tetrahymena pyriformis W. Nor were they mutagenic or sensitizing. At the same time, PPCs showed pronounced adaptogenic and growth-promoting activities.

[Architectonics of Bifidobacteria populations: submicroscopic aspect of cell cohesion in Bifidobacterium adolescentis and Bifidobacterium bifidum]. / Arkhitektronika populiatsii Bifidobakterii submikroskopicheskii aspekt kogezii kletok Bifidobacterium adolescentis i Bifidobacterium bifidum.

Populations (cultures) of Bifidobacterium adolescents and B. bifidum, growing on artificial liquid and agar media, are presented by highly ordered mycelial structures. The topography of them depends on mutual arrangement of polymorphic cells and the way of their daughter cells separation after division. Evidences obtained by scanning electron microscopy (SEM) of total preparations and by transmission electron microscopy (TEM) of ultrathin sections correlate well. These data showed the existence of morphologically varied intercellular contacts (coherence) that ensure the stability of such microbial consortia during adaptation to ambient conditions. Intercellular contacts with the aid of different extracellular structures--microfibrillae, knob-like juts, cell wall evaginations, and capsuleform stuff(glycocalix)--are the result of genetically determined self-regulating development of microbial populations as multicellular systems.