Quantitative method for the determination of antibiotic-resistant gut bacterial strains for the early diagnosis of chronic arthritis.

Based on the clinical and microbiological monitoring of two groups of children aged 3 to 17 years with acute (n=78) and chronic (n=46) course of reactive arthritis (ReA), a method for early diagnosis of chronic arthritis was developed by determining the number of antibiotic-resistant coprostrains in patients with ReA, characterized by the absence of the need to isolate a pure culture of the pathogen and its identification; inoculation of faeces at a dilution of 10-5 on solid 1.5% GRM-agar with an antibacterial agents used in the treatment of a particular patient, at a minimum inhibitory concentration in the resistance range, followed by incubation and counting of the colonies of microorganisms grown on the plate. A significant relationship between the number of antibiotic-resistant gut bacterial strains and the course of arthritis (acute, chronic) was revealed, and the borderline value of the number of antibiotic-resistant gut bacterial strains was determined - 5×103 CFU/g, which allows differentiating the acute course from the chronic one: in the acute course< 5×103 CFU/g, with chronic - ≥ 5×103 CFU/g. The method allows, at the stage of completion of anti-inflammatory therapy in the active phase of the disease, to identify a risk group for the development of a chronic course of arthritis among patients with ReA, which can contribute to timely therapeutic measures aimed at preventing recurrence of the disease and making the patient disabled.

Diagnostic cytokine marker of male infertility - interleukin 4.

Analysis of the study is to assess the diagnostic significance of cytokines in the sperm plasma of men of reproductive age (20 - 45 years) of two groups: of patients with chronic bacterial prostatitis, not complicated by infertility and with loss of fertility. The study of sperm plasma - the WHO standard. Determination of the level of cytokines in seminal plasma - by enzyme immunoassay («Cytokine¼, Russia). Two methods of mathematical statistics were used: discriminant analysis and classification trees (decision trees).The similarity of interpretations of discriminant analysis and decision tree was noted, where the main role in both cases belongs to the cytokine IL-4. The level of sperm IL-4 in combination with therapeutic monitoring can be used for the medical management of patients with chronic prostatitis in order to prevent the development of infertility and to develop methods for screening diagnostics of fertility disorders in men.

[METABOLIC PROFILE OF BIFIDOFLORA UNDER DIFFERENT MICROECOLOGICAL CONDITIONS OF THE COLON BIOTOPE IN HUMAN].

AIM: To study the spectrum and level of short-chained fatty acids (SCFA) in supernatant of bifidobacteria under different microecological conditions ofthe colon biotope in human. MATERIALS AND METHODS: Metabolites of 88 bifidobacteria strains isolated from patients when examined for dysbiosis of the colon were investigated. Definition of concentration of SCFA was performed on acidified supernatant samples by a separation method on chromatograph GC-2010 Plus, Shimadzu (Japan). RESULTS: Monobasic acids were found in metabolites of 50 - 100% study cultures of bifidobacteria where the spectrum and level of carboxylic acids in supernatants varied depen- ding on microecological condition of the origin of the discharge. In severe damages of microsymbiocenosis in metabolites of Bifidobacterium spp., summarized concentrations of SCFA, structural index, levels of aceitic and propionic acids were decreased. Strain-specific differences in a metabolic profile of bifidofloia in a composition of individual consortiums were determined. Data obtained indicate the variation of functional (metabolic) activity of dominant strains in different microecological conditions of the human colon. CONCLUSION: Uniquieness of metabolome of every other strain due to their strain specifity determines their functional activity, but a metabolic profile of bifidoflora can serve as the most important criterion for the selection of effective probi- otic drugs for treatment and prevention of dysbioisis in the colon.

[GUT MICROSYMBIOCENOSIS IN CHILDREN WITH REACTIVE ARTHRITIS].

AIM: To study the state of gut microsymbiocenosis in children with reactive arthritis (RA), with the assessment of biofilm formation (BFF) of microsymbionts and the ability to change cytokine levels (their anticyokine activity) in vitro. MATERIALS AND METHODS: The investigation of gut microsymbiocenosis by means of bacteriological method was conducted in 34 children with RA and 25 relatively healthy 3 - 16 year- old children. Microorganisms were identified with the help of MALDI-TOF mass-spectrometry, anticytokine activity (ACA) of microsymbionts - according to Bukharin O.V et al. (2011), biofilm formation - according to O'Toole G.A., Kolter R. (1998). RESULTS: On the ground of species composition differences of gut microbiota discrimination model was created which allowed to separate the group of children with RA from healthy individuals. Microsymbiocenosis of patients with RA was characterized by increasing number of opportunistic microorganisms (OM) (enterobacteria, clostridia, bacteroides, and Candida), BFF and ACA level. CONCLUSION: The obtained data greatly contribute.to the deciphering of spondylo- arthritis and disclose the role of microbial factor under given pathology. Hypercolonisation of human gut with OM, having pronounced ability to BFF and regulating cytokine level, promotes strengthening of arthritogenic potential and serves as additional marker of arthritis development risk in children.

[IMMUNE REGULATORY PROPERTIES OF BIFIDOBACTERIA METABOLITES DURING EUBIOSIS AND DYSBIOSIS OF THE HUMAN COLON].

AIM: Evaluate immune regulatory properties of bifidobacteria metabolites during eubiosis and dysbiosis of the human colon. MATERIALS AND METHODS: Anti-cytokine activity of metabolites of bifidobacteria clinical strains and their ability to influence the production of pro- and anti-inflammatory cytokines (IL-10) by peripheral blood mononuclear cells of healthy humans was studied, taking into account microecological state of the human intestine. Determination of final concentration of cytokines in experimental and control samples was carried out by EIA. RESULTS: Sensitive parameters, that are suitable for evaluation of stability of human intestine microsymbiocenosis, were detected. The level of microbial seeding, concentration of TNF-α and anti-lysozyme activity turned out to be informative for bifidobacteria in eubiosis conditions. The ability of bifidoflora metabolites to influence the production of pro-inflammatory cytokines (INF-γ, TNF-α, IL-8) by human mononuclears was a significant parameter during formation of 1 - 3 degree dysbiosis. CONCLUSION: The maintenance of physiological state of intestine homeostasis is determined by immune regulatory properties of bifidobacteria metabolites, that is realized via their interaction with both cytokines (anti-cytokine activity) and production of cytokines by host immune cells (peripheral blood mononuclears).

[THE ROLE OF BIFIDOBACTERIA IN THE FORMATION OF HUMAN IMMUNE HOMEOSTASIS].

In the review the materials on the formation of intestinal immune homeostasis through involvement of bifidobacteria which are the key species of microbiota of human colon biotype are presented. Key function of dominant microorganisms, bifidoflora in particular, in intestinal biotype of a host is carried out by means of maintenance of self microorganisms and pronounced antagonism concerning non-self. Realization of this principle in intermicrobial relations allowed to develop algorithm of microbial self-non-self discrimination in microsymbiocenosis on the basis of detected opposite phenomenon (enhancement/suppression) of the main physiological functions of microsymbionts survival (reproduction and adaptation) in dominant-associant pair. Primary discrimination of foreign,material by bifidobacteria is the initial stage of the following "signaling" in the regulation of host immune homeostasis. Further stages of regulation occur by activation of dendritic cells by bifidobacteria with the sequential influence on differentiation of Th0 towards regulatory lymphocytes. The formation of Treg and regulation of immune homeostasis are carried out by bifidobacteria: due to direct activation of dendritic cells (ligand-receptor interactions) and maintenance of optimal cytokine balance.

[Mechanisms of realization of protective effect of miliacin during experimental Salmonella infection: effect of endotoxinemia and cytokine production].

AIM: Evaluate the effect of miliacin on the intensity of endotoxinemia and features of cytokine production in experimental salmonella infection. MATERIALS AND METHODS: The studies were carried out in 128 male mice (CBAxC57Bl6)F1 divided into 4 groups: I--intact; II--infected; III--infected after administration of miliacin solvent: tween-21; IV--infected after administration of miliacin. Determination of the endotoxin in blood plasma was carried out by using chromogenic LAL-test. Cytokine production was studied in splenocyte culture by EIA method. RESULTS: Miliacin reduced the intensity of endotoxinemia in mice of group IV. Salmonella infection increased spontaneous (IFNγ) and induced (IL-12, IFNγ, IL-17) cytokine production. Miliacin ensured the most significant increase of spontaneous IL-10, IL-12 and IFNγ production compared with groups II and III. At the same time it limited the increase of induced IL-17 production compared with groups II and III. CONCLUSION: Protective effect of miliacin is determined by the reduction of endotoxinemia, mobilization of Th-1 response, stimulation of IL-10 production and limitation of IL-17 participation in the development of the inflammatory reaction.

[Protective effect of miliacin during experimental salmonellosis infection].

AIM: Evaluation of influence of triterpenoid miliacin on the development of experimental salmonellosis infection. MATERIALS AND METHODS: Studies were carried out in 330 male mice (CBAxC57Bl6)F1. Miliacin was administered 3 times intraperitoneally with the interval of 3 days between administrations at a single dose of 2 mg/kg. The animals were infected intraperitoneally by hospital origin Salmonella enteritidis strain (2x10(6) bacteria per mice). 4 groups of mice were used: I - intact; II - infected; III - infected after administering solvent for miliacin 3 times (tween 21 at final concentration of 1.6x 10(-7) mol/kg); IV - infected after administration ofmiliacin. RESULTS: Miliacin reduced the mortality of mice compared with groups II and III. Microbial contamination of mice spleen in group IV was significantly lower compared with group II at all the periods of the study, and liver - at days 10 and 15. Triterpenoid weakened cell depletion of bone marrow, thymus and limited hyperplasia of spleen compared with animals of groups II and III. Its protective effect did not correlate with increase of antibody titers. CONCLUSION: Miliacin weakens the severity of salmonellosis infection course.

[Risk factors for violations of the fertility in men living in urban areas].

The immunocytokine status of the reproductive tract was evaluated in men with different forms of gonorrhea, who lived in the urbanized areas of the Orenburg Region. The typical form of gonorrhea, which is a risk for infertility, is accompanied by the reduced levels of lysozyme and IL-10 and the increase content of lactoferrin and proinflammatory cytokines, such as IL-1, Ibeta-6, and IL-8 in the ejaculate.

[Resistance of the fungi Candida to human innate immunity factors].

The paper presents the results of studying the anti-lactoferrin and sIgA-protease activities of Candida strains isolated in varying human intestinal dysbiosis. Thirty-eight Candida strains were isolated from patients with dysbiosis. The isolated strains were found to have anti-lactoferrin and sIgA-protease activities. The wide spread of the examined properties of Candida and their dependence on its species and the degree of dysbiotic disorders were established. The findings gain more insight into the range of biological properties in the fungi Candida, which contribute to the long-term survival of their host with microenvironmental abnormalities.

[Features of immunity in tobacco smoking among adolescents].

The work is devoted to the study of indicators of systemic and local immunity in the respiratory tract in healthy adolescent tobacco use. The aim of the work is to characterize indices of general and local immunity of the respiratory tract during smoking in healthy adolescents. The study of local immunity was performed in induced sputum with the use of 0.1% solution of dithiothreitol. The study used tests assessing immune status I and II level. Active smoking in healthy adolescents induces a local and general immunological signs of inflammation: in sputum--elevation of IL-1alpha level and number of cells with activation markers (SD45 and HLA-DR); in the peripheral blood--the rise of levels of IL-6, IL-8, TNF CD19 (+) - cells, IgE and IL-4 in combination with a reduction in the number of CCD3(+)-, CD4(+)-, CD8(-)-lymphocytes and depression of the phagocytic activity of neutrophils.

[Species characteristic and persistent factors of Candida genus fungi isolated from various biotopes during infectious-inflammation diseases and intestine dysbiosis].

AIM: Determine species composition and persistent properties of Candida genus fungi isolated from various biotopes of the human organism during infectious-inflammation diseases and intestine dysbiosis. MATERIALS AND METHODS: 152 Candida genus fungi were isolated and identified from individuals with dysbioses and patients with infectious-inflammation diseases. Antilactoferrin and sIgA-protease activity of isolates was determined by enzyme immunoassay. RESULTS. C. albicans strain was shown to be the dominant species isolated from all the studied biotopes. Wide prevalence of the studied properties in Candida genus fungi and their dependence on species membership, isolation biotope, infectious process form, degree ofdysbiotic disorders in intestine was established. CONCLUSION: The data obtained expand the understanding of biological properties of Candida genus fungi that facilitate their prolonged survival in host organism.

[Anti-cytokine activity of microorganisms].

AIM: Development of a method of determination of anti-cytokine activity (ACA) of microorganisms, study of the prevalence and intensity of ACA to pro- and anti-inflammatory cytokines in pathogenic and opportunistic bacteria. MATERIALS AND METHODS: ACA was determined in 72 strains of microorganisms including members of the intestinal microflora and strains of pathogenic bacteria (salmonellae andgonococci). Study of the ability of supernatants and live cell cultures of microorganisms to induce changes in pro- (IFN-gamma, TNF-alpha, IL-6) and anti-inflammatory cytokines (IL-4, IL-10) was performed by using co-incubation of exometabolites and live cell bacteria, fungi with recombinant cytokines. RESULTS: Amethodological approach allowing the determination of ACA, the prevalence of which among studied microorganisms was 50 - 62%, was developed. A decrease of cytokine concentration in the medium was registered in co-incubation of them with supernatants (in 56% of cases) and to a lesser degree--with live cell cultures (44%) of the studied bacteria and fungi. Expression ofanti-cytokine activity was the most pronounced to TNF-alpha (Bifidobacterium spp. and Lactobacillus spp., Neisseria gonorrhoeae), IFN-gamma (N. gonorrhoeae and Salmonella enterica) and IL-10 (Bifidobacterium spp. and Lactobacillus spp.). CONCLUSION: The data obtained expand the conception of modification of cytokine dynamic by pathogenic and opportunistic microorganisms, that can influence the course and outcome of an infectious process.

[Characteristic of biological properties of Neisseria gonorrhoeae and parameters of local immunity during urogenital gonococcal infection].

AIM: To study the prevalence and expression of antilactoferrin, IgA- and slgA-protease activity of gonococci and state of local immunity during various forms of urogenital gonorrhea. MATERIALS AND METHODS: Ability to inactivate lactoferrin (ALfA), IgA and secretory IgA (slgA) was studied in 28 Neisseria gonorrhoeae strains isolated from patients with localized gonorrhea and 26 strains isolated from patients with systemic signs of gonorrhea. State of the local immunity was assessed on the lactoferrin, IgA and slgA levels, which were measured by immunofluorescence assay in ejaculate of 54 patients with gonorrhea and 18 healthy males. RESULTS: Penetrance of ALfA, IgA- and slgA-protease activity of gonococci did not depend from form of infection. Expression of studied characteristics of gonococci as well as combination of ALfA and slgA-protease activities were more prominent in patients with systemic signs of gonorrhea. The same patients had higher level of lactoferrin in semen and, in contrast, lower levels of IgA and slgA compared with patients with localized gonorrhea. CONCLUSION: Strains of gonococci inactivating lactoferrin, IgA and slgA depress mucosal barrier of urogenital biotope and create conditions for the development of disseminated forms of gonococcal infection.

[Phenotypic characteristic of staphylococci and local immunity during bacterial carriage].

AIM: To assess prevalence and intensity of antilactoferrin and slgA-protease activity in strains of staphylococci and to study the state of local immunity in resident and transitory bacterial carriers. MATERIALS AND METHODS: Strains of Staphylococcus aureus and S. epidermidis isolated from 67 healthy persons, who were tested for staphylococcal carriage with subsequent differentiation of types of carriage according to Chistovitch's method, were studied. Antilactoferrin and slgA-protease activity as well as levels of lactoferrin and slgA in nasal secretions were determined using fluoroimmunoassay. RESULTS: Higher prevalence as well as intensity of antilactoferrin and slgA-protease activity and combination of these characteristics was determined for strains of S. aureus from resident carriers compared with strains from transitory carriers. Higher levels of lactoferrin and slgA were observed in nasal secretions of resident carriers compared with transitory carriers. CONCLUSION: Studied factors could be the markers of resident type of carriage that should be considered for the development of effective measures for sanation in persons with prolonged bacterial carriage.

[Interaction of a causative agent with associative bacteria during salmonellosis].

AIM: To determine a composition of gut microflora during salmonellosis and to study the modification of persistent characteristics (antilysozyme activity, ALA) of symbiotic microorganisms in associations. MATERIALS AND METHODS: Bacteriologic study of feces was performed in 90 patients aged 18-39 years, which were divided to three groups: patients with salmonellosis in acute phase, reconvalescent patients, and conditionally healthy persons. Condition of gut microflorawas determined; microorganisms associated with Salmonella infection were isolated, and their influence on ALA of Salmonella was studied. RESULTS: Gut microbiocenosis was more diverse in patients compared with healthy persons. Significant reduction of bifidobacteria quantity (to 10(7) CFU/g of feces and less), especially in reconvalescent period, was noted. Association between bifidoflora deficiency and excessive increase of quantity of yeast fungi was revealed. It was determined that exometabolites of indigenous anaerobic microflora (bifidobacteria) promoted decrease of ALA of Salmonella, whereas opportunistic facultatively anaerobic microorganisms (enterobacteria, staphylococci) rendered mainly stimulating effect on the ALA of Salmonella. CONCLUSION: Obtained data reveal characteristics of bacterial interactions in associative symbiosis and provide insights about mechanisms of formation of pathobiocenosis and state of bacterial carriage.

[Algorithm for prediction of reconvalescent salmonellosis carriage].

Of total of 181 patients with Salmonella infection were examined by clinical and laboratory methods. As many as 124 indices characterizing biological properties of Salmonellae, a large intestine microbiocenosis status, systemic and local immunity, were investigated as well. Among these indices, the most revealing signs were defined to prognosticate the reconvalescent carrier state. Conclusion was made on the prevalence of immune mechanisms in the development of reconvalescent salmonella carriage and on the important role of the ability of a causative agent to inactivate the factors of host innate immunity in this process. Diagnostic prognosis algorithm for Salmonella bacterial carriage was worked out. Clinical testing confirmed its effectiveness.

[Influence of plasmid pR50 controlling bacterial protease activity on Salmonella experimental infection].

The comparative analysis of the infectious process and immunological parameters in (CBA x C57BL/6)F1 mice infected with S. typhimurium isogenic strains differing by the presence of plasmid pR50 determining protease activity, was carried out. A growth in the expression level of antilactoferrin, anticomplementary and anti-immunoglobulin activity in bacteria isolated from the spleen in the course of the infectious process was detected. In mice infected with S. typhimurium having plasmid pR50, in contrast to nonplasmid recipients, a higher level of contamination of organs, the suppression of spontaneous, stimulated production of interferon-gamma and the bactericidal properties of peritoneal macrophages were noted. The data obtained in this investigation suggested that the acquisition of R-plasmid of 50 MD, controlling protease activity and multiple medicinal resistance, contributed to the persistence of intracellular bacteria.

[Biological properties of salmonellae and local immunity in Salmonella infection].

The comparative study of the biological properties of S. enteritidis and S. typhimuruim, isolated from patients and convalescent carriers, was carried out. Factors inactivating the components of the local immunity of the intestine (lysozyme, complement, lactoferrin, IgG, IgM and IgA) were detected in the causative agents of Salmonella infections. The spread and expression degree of properties of a causative agent were serovar-depended: high penetration characteristics and the expression of anti-lactoferrin and anti-immunoglobulin activity were characteristic of S. typhimurium. S. enteritidis strains isolated from patients with carrier state formed in the convalescence period were found to have higher persistence level. In co-profiltrates obtained from carriers at the peak of the disease and during convalescence lower levels of IgM, IgG, sIgA, complement and lactoferrin were determined in comparison with those in coprofiltrates obtained from patients in whom no subsequent carrier state was formed. These results indicate that an increase in the persistence of salmonellae, occurring simultaneously with the local immunodeficiency, contributes to the prolonged survival of bacteria in the intestine.

[New method for the determination of anti-lactoferrin activity of microorganisms]. / Novyi metod opredeleniia antilaktoferrinovoi aktivnosti mikroorganizmov.

A new method for the determination of the antilactoferrin activity (ALfA) of microorganisms, based on the detection of lactoferrin by the enzyme immunoassay, is proposed. The new method widens the spectrum of microorganisms to be tested, makes it possible to detect lactoferrin inactivation by bacteria producing antagonistically active substances (muramidases, organic acids, hydrogen peroxide, etc.), increases the reliability of the determination of the ALfA antilactoferrin activity of microorganisms due to the increased accuracy of its quantitative characterization. Testing of the culture fluid for lactoferrin following the growth of microorganisms in a medium with this protein revealed the capacity to inactivate lactoferrin in bacteria (Escherichia coli, Salmonella sp.) and yeast-like fungi (Candida sp.), isolated from feces and clinical material, of persons with carrier states, dysbiotic disturbances and pyoinflammatory diseases.