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Annually, data are accumulating on the involvement of opportunistic microorganisms in the development of inflammatory diseases in humans, maintaining a chronic inflammatory response and, thus, adapting to the conditions of existence in the biotopes of the human body. This review provides information on the interactions of microorganisms of medical importance, which affects the virulence of both opportunistic pathogens and classical pathogens, which probably underlie the chronicity of infection and inflammation. Often, opportunistic pathogenic species cannot fully realize their pathogenic potential, which is observed in numerous cases under conditions of microbial symbiosis. Thus, a revision of approaches to interpreting the results of microbiological methods is necessary, which takes into account the functional activity of the total microflora and the search for individual extrachromosomal genetic elements as a marker of the pathogenicity of microorganisms.

Introduction. Currently, there is an increasing importance of microbial associations in the pathogenesis of genital inflammatory diseases. However, the issues of deciphering the taxonomic affiliation and the diagnostic significance of the bacteria detected in this case remain unresolved.The aim of the study was to review the diagnostic significance of the quantitative approach in determining the etiological role of microorganisms in andrology.Materials and methods. For the study, samples of ejaculate and/or discharge from the urethra from 15 men who were in infertile marriages, 12 with a confirmed diagnosis of "acute genital gonococcal infection" were used. The testing included a classic bacteriological study and the metagenomic analysis of 16S ribosomal RNA samples carried out at the Department of Collection Cultures of the State Research Center for Applied Microbiology (Obolensk). To standardize the distribution of samples into groups based on the indicators of alpha diversity and the concentration of putrescine, the range of variation and the average linear deviation were used. Statistical analysis of the obtained data was carried out using Excel software (MS Office package).Results. The microbial landscape of the ejaculate was characterized by the predominance of representatives of the genera Enterococcus, Neisseria, Lactobacillus, Corynebacterium, Prevotella, Bacteroides. Stable associations of E. faecalis and M. osloensis were detected in the ejaculate. When using the cultural method, representatives of the genus Moraxella were not isolated in any sample. It was shown that discrepancies can affect not only quantitative indicators, but also reveal inconsistencies between the qualitative assessment of detected genetic markers and the results of identification in a bacteriological study of individual representatives of similar or phenotypically similar taxa.Discussion. The results of this study indicate that the lesser diversity of opportunistic pathogens gives them more opportunities to realize their pathogenic potential. On the other hand, in a complex community with greater alpha diversity, its realization is hindered by a complex intermicrobial relationships and the need to survive.Conclusion. As a result, it seems that the most promising approach is the integrated use of cultural methods and metagenomics with a comparative statistical analysis of the obtained qualitative and quantitative indicators.

Introduction. Currently, the colonization of various human biotopes by yeast-like fungi of the genus Candida is considered a relatively frequent phenomenon. At the same time, the clinical manifestations of the inflammatory process do not develop in every case, which implies the formation of a unique symbiosis between microscopic fungi and cells of the human body, the maintenance of which largely depends on the activity of the immune system. The main part of researches on the antifungal activity of the human immune system is concentrated around pathological conditions, and practically no attention is paid to such in healthy individuals. It has been shown that human immunity factors can, on the one hand, for example, contribute to the formation of C. albicans biofilms, and, on the other hand, take an active part in their destruction.The aim of the investigation was to evaluate the candidacid, antibiofilm, phagocytic and radical-producing activities of peripheral blood of healthy donors using C. albicans cells as an object.Materials and methods. Peripheral blood samples were obtained from 32 healthy donors, mycidal activity, absorption and radical-producing abilities of leukocytes, as well as the effect of blood serum on film biomass were assessed. For opsonization of C. albicans cells, immunoglobulins G were used according to the previously approved method.Results. A weak mycocidal activity of the peripheral blood of healthy donors was shown. Opsonization of C. albicans with immunoglobulin G significantly increases this blood function. In the early phase of contact with C. albicans, yeast-like cells mainly absorb by neutrophilic leukocytes, and mononuclear cells practically do not participate in the process of phagocytosis, probably their activity manifests itself in a later period. Opsonization of C. albicans stimulates the absorption activity of leukocytes, which is reflected in an increase in the average number of absorbed objects per leukocyte. It has been shown that opsonins can participate in enhancing the radical-producing activity of leukocytes. Thus, inactivation of proteins of the complement system levels the stimulating effect of C. albicans opsonization.Conclusion. Immunoglobulins G and proteins of the complement system make a significant contribution to the suppression of the pathogenic activity of C. albicans.

Currently, in the diagnosis of diseases, a decisive place is given to laboratory methods, which should be informative, relatively simple to perform and rapid. The article describes the approbation of a method for rapid detection of Escherichia coli and bacteria of Escherichia coli group in the oral cavity. Research involved 44 volunteers, who were sampled from the oral cavity, followed by incubation in Koda’s medium. The study used oral (n=11) and gingival fluids (n=11); smears-prints from the oral mucosa (n=11); dental biofilm (n=11). After 24 hours, the change in color and transparency of the medium was assessed. The preservation of the initial green color and transparency by the medium meant the absence of E. coli and bacteria of Escherichia coli group in the sample. A change in the color of the medium to yellow, turbidity and / or the formation of bubbles indicated the presence of E. coli and bacteria of Escherichia coli group. In parallel, the material was inoculated onto Endo agar, followed by identification of strains to species. As a result of the study, a complete coincidence of the results of the classical bacteriological method and the method using Koda medium was shown. In the latter case, a significant advantage is the speed of obtaining the result (18-20 hours), in contrast to the classical method, the interpretation of the results of which is available only after 72 hours or more. All of this is in line with the state of the art in clinical microbiology and rapid diagnosis based on «point-of-care testing / doctor’s office» diagnostic principle. The presented method can be successfully applied in clinical practice for topical diagnosis of microorganisms E. coli and bacteria of Escherichia coli group in the oral cavity.

Introduction. At present, the question of commensal, including opportunistic, microflora participation in infertility development remains debatable. In a number of studies, the translocation of the vaginal microflora into the endometrial tissue is considered as a factor contributing to inflammation development. In addition, the connection of some reproductive losses with the persistence of certain conditionally pathogenic microorganisms is shown. Today, to solve this issue, molecular genetic research methods are being actively introduced that surpass the routine cultivation techniques in a number of positions.The aim of the study was to assess the taxonomic diversity of microorganisms in the vaginal biotope with infertility.Material and methods. For the study samples of vaginal contents obtained from the posterior vaginal fornix of 15 women, consisting of barren marriage, were used. A metagenomic study of 16S ribosomal RNA samples was carried out on the Illumina MiSeq platform, using the MiSeq Reagent Kits v3 kit (600-Cycle Kit), as recommended by the manufacturer. Libraries for sequencing plots of the V3-V4 gene of the 16S ribosomal RNA were prepared according to the 16S Metagenomic Sequencing Library Preparation Illumina. In bioinformatics assessment, Kraken Metagenomics version 2.0.0 software for metagenomic analysis (classifier of reads — short nucleotide sequences) was used using a standard database.Results. It was shown that the occurrence of representatives of the Lactobacillaceae family in the vaginal biotope varies from 12 to 84%. The genus Lactobacillus with the dominance of L. jensenii, L. delbrueckii and L. amylolyticus occupied the leading position among the members of the family. In all samples, Moraxella spp. was found in large numbers, with M. osloensis leading among the representatives of the entire community. In half of the cases, the joint presence of M. osloensis and G. vaginalis was revealed.Conclusion. It has been established that in infertility in the vaginal microbiota the number of representatives of the genus Lactobacillus is significantly reduced, and there is also a change in the leading species to L. jensenii, whose functional activity does not fully ensure the colonization resistance of the vaginal biotope, allowing for excessive reproduction of oppotrunistic microorganisms, in particular, M. osloensis.

Microbial biofilms are heterogeneous, moving and constantly changing communities of microorganisms, often of various taxons. Approaches to study and assessment anti-biofilm drugs widely available today do not adequately assess their effects, while the results of studying the interaction of drugs with components of the film composition can provide them the right choice. The aim of investigation was to test a new method of morphological evaluation of biofilms. To form biofilms, we used an approach when the slide was placed at an angle of 30o-45o relatively to the Petri dish, and a suspension of test strains S. epidermidis in peptone broth was poured into the space between the Petri dish and the slide. A sterile cotton swab moistened with distilled water was placed next to the glass slide to create optimal humidity. The system was placed in a thermostat for 24 hours. The formed films were examined under a microscope using the DCM 310 video eyepiece and the Scope photo x86,3.1.312 program that allowed to conduct a complete morphometric study of the film: select layers, channels, cavities and make measurements, and then save the results on electronic media in jpg file format. Microscopy of the stained slides revealed that the biofilm has a layered structure. In each image obtained using a video eyepiece, it was possible to differentiate 4 layers. From the border of the two media to the inside: the fragmentation layer, the dense layer, the matrix substance layer, and the last one - the persistence layer. Channels of different diameters (from 10 to 24 microns) are observed across the entire thickness of the biofilm. Thus, used approach allows us to visualize and evaluate the structure of microbial biofilm, measure the thickness of layers and channel diameters. In addition, this method can be used to study the effect of antimicrobial drugs on bacterial films.

Microorganisms are able to form biofilms on surfaces of biotic and abiotic nature. In turn, in human biotopes there are optimal conditions for the implementation of biofilm-forming activity. Moreover, in medical practice, polymeric materials are often used for drainage or prosthetics, which can also be successfully colonized by bacteria. However, in laboratory practice, the formation of biofilms is usually evaluated on glass or polystyrene. The purpose of the study is to evaluate the methodological features of studying the biofilm-forming activity of microorganisms on the surface of synthetic polymeric materials. We used strains of Staphylococcus aureus ATCC 25923, Escherichia coli K-12, Candida albicans ATCC 10231, as well as synthetic polymeric materials - DentLight Flow light-curing composite material (nano-hybrid fluid composite; Russia), glass ionomer chemical curing Fuji 1 (Japan), cement for temporary fixation of orthopedic constructions TempBond NE (USA), acrylic, polyurethane and polyvinyl chloride. The formation of biofilms in flat-bottomed ELISA plates in this study was considered as a control group. If the polymer belonged to cold curing materials, sterile flat-bottomed tablets were used, the bottom of which was filled with a thin layer of plastic. After hardening of the plastic, biofilms were formed in the tablets. In the second series of experiments, hot cured materials cut into equal parts 5×5×1 mm in size were placed in the wells of a plate and again used to determine biofilm formation with subsequent coloring. To extract the dye, the pieces were transferred to a new plate to exclude the amount of film biomass formed on the walls of the plate wells. In both cases, cultivation was carried out at 37° C for 24-48 hours. The biomass of the film was stained with fuchsin. Statistical data processing was performed using t-Student criterion. For the threshold level of significance, the value p

Nowadays, in the presence of wide diagnostic possibilities, laboratory diagnostics of microecological disorders of vaginal biotope are often limited to clinical data, microscopic examination results and the use of a culture method. However, with such a complex it is impossible to get an answer about the functional activity of microorganisms. The aim of investigation was to evaluate the information content of a combined study of growth parameters and the ability to produce lactic acid by clinical strains of Lactobacillus spp. to characterize the state of the microecology of the cervical-vaginal biotope. Materials and methods. Studied the growth kinetics of strains of lactobacilli isolated from the detachable posterior vaginal fornix. The concentration of lactic acid in the medium was determined using the “Lactic Acid - Olvex” kit (Russia). Samples were taken every 12 hours of cultivation. Statistical analysis of the results was performed using the methods of descriptive statistics, Student’s t-test. Results. It has been shown that 44% of Lactobacillus spp. to 72 hours of cultivation did not reach the phase of exponential growth. The remaining strains from 12-24 hours passed into the phase of exponential growth. In the production of lactic acid, the strains that are in the lag-phase did not differ from each other, since they practically did not synthesize this metabolite. Among the remaining strains that are in the phase of exponential growth, some did not produce lactic acid, others increased their lactate production every hour. Part of the strains reached the peak of acid production by 36 hours and by 72 hours some decrease in synthetic activity was observed. Conclusion. It has been shown that for most vaginal strains of Lactobacillus spp. characteristic variability of the duration of the adaptation period and the level of functional activity. In addition, only a small number of clinical strains produced lactic acid 24 hours after the start of cultivation. Therefore, to adequately assess the microecological status of the vaginal biotope, microscopic examination of both native material and cultures isolated on special nutrient media is not enough. It seems that, along with the use of modern methods of genetic analysis, the determination of in vitro growth characteristics, primarily lag-phase duration, and lactate production by lactic acid bacteria strains can clarify many issues related to the formation of dysbiotic states, in particular, in the vaginal biotope, and will also serve to increase the effectiveness of the prescribed treatment.

Despite the long history of the study, laboratory diagnosis of gonococcal infection remains a complex task that does not have a clearly regulated effective solution. Aim of investigation was to assess the species diversity of the microbiota of the genital tract of men and women with suspected acute genital gonococcal infection (AGGI) using test systems of Russian manufacturers. A study of samples of the contents of the urethra of 69 men and posterior vaginal fornix fluids of 33 women of reproductive age with characteristic clinical manifestations and a presumptive diagnosis of AGGI was made. Cultivation was carried out using elective culture media with subsequent identification of strains by biochemical properties. Detection of DNA of Neisseria gonorrhoeae, Staphylococcus spp., Streptococcus spp., Enterobacteriaceae, Gardnerella vaginalis, Atopobium vaginae, Lactobacillus spp. performed by PCR using Vektor-Best and InterLab Service kits (Russia). All patients were divided into groups according to the results of the bacteriological method and PCR. A metagenomic study of 16S ribosomal RNA samples was performed on the Illumina MiSeq platform using the MiSeq Reagent Kits v3 kit (600-Cycle Kit). Statistical analysis of the data was performed using criterion x2. As a result of a laboratory study, the presumptive clinical diagnosis of «AGGI» found its bacteriological confirmation in 35.3% of cases only, among which fragments of the N. gonorrhoeae genome were detected in 63.9% of the samples only. Moreover, a wide variety of microorganisms in the genital tract of both men and women was found in metagenomic analysis. However, this technique does not allow us to assess the viability of the detected bacteria, and the microflora spectrum is excessively wide. In addition, the high level of genetic polymorphism of different strains of N. gonorrhoeae complicates the interpretation of the results. Deciphering the composition of microbiota allows the use of InterLab Service kits. The decoding of the etiology of purulent-inflammatory processes in the genital tract, which presents serious difficulties, is greatly facilitated by the use of Russian kits for molecular genetic analysis, which, in our opinion, provide the necessary and sufficient information for practice.

Current methods of biofilm imaging do not support a differentiated assessment of its composition, since it is not possible to establish a substrate stained with crystal violet, as this dye can form complexes with both intracellular and extracellular structures. This approach does not adequately assess the anti-biofilm effects of drugs, while the results of studying the interaction of drugs with biofilm components can ensure their most correct choice. The aim of investigation was to study the possibility of applying the original modification of the current method to determine the ratio of the cellular part and the matrix of biofilms of gram-positive microorganisms. The biofilm components were analyzed using a two-step approach, when prepared biofilms of gram-positive microorganisms were stained with crystal violet for 5 minutes, followed by fixing the dye in bacterial cells with iodine solution, and then the colored products were dissolved with 95% alcohol: matrix components for 1 minute, total biofilm for 15 minutes, after which the composition of biofilms was estimated by the formula: M=(OP1/OP15)×100, Kb=100-M, where M is the proportion of the matrix,%; Kb - the proportion of the cellular component,%; OP1 - optical density of samples, when alcohol was allowed to dissolve the colored product for no more than 1 minute; OP15 - was the optical density of samples, when alcohol is allowed to dissolve the colored product for 15 minutes. It was shown that in the composition of the biofilm formed by the collection strain, the proportion of the matrix was 13.2%, and the cellular component accounted for 86.8%. When the same strain cultivated in the presence of an antibiotic, an increase in the biofilm matrix was observed, which is probably due to the compensatory response of the microorganism to the action of the antibiotic. The proposed approach to the study of biofilms makes it possible to evaluate its component composition. Obtaining additional information in this way can provide, inter alia, an increase in the effectiveness of antimicrobial therapy while reducing the study time.