[Etiology of purulent-inflammatory processes in the genital tract: suspicions of clinicians and problems of laboratory confirmation.]

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 «AGGI¼ 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.

[The determination of biofilm composition of gram-positive bacteria.]

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.

[Characteristics of lactobacteria strains, having diagnostic significance in gynecological practice.]

BACKGROUND: 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. CONCLUSIONS: 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.

[GUT AND VAGINAL MICROBIOTA IN FEMALE WITH REPRODUCTIVE DISORDERS ASSOCIATED WITH GASTROINTESTINAL DISTURBANCE].

The aim of research - a comparative study of the microflora of the vagina and bowel in women with secondary infertility and gastrointestinal distress. Materials and methods. We examined gut and vaginal microbiota in 17 infertile women according to the presence or absence of gastrointestinal diseases. We used a standard procedure for microbiological examination. Results. Certain changes of bacterial load and composition in both reservoirs were established. They allowed us to conclude the same trends in characterizing of the developing dysbiosis. Conclusion. Thus, the simultaneous studying of vaginal and gut microflora seems to be rational during the examination of women with childbearing disorders. It could possibly increase the treatment efficacy and/or prevent the development of some pathology.

Cell-Mediated Hemolytic Activity of Nosocomial Pseudomonas Aeruginosa Strains.

Cell-mediated hemolysis and adhesion index of nosocomial P. aeruginosa strains were experimentally studied. The highest hemoglobin release was recorded after centrifugation of erythrocyte and bacterial cell suspension preincubated at 37 C. All cultures were referred to highly adherent variants. The relationship between P. aeruginosa adhesion activity and erythrocyte lysis was found only in "passive" cell-cell contact. No correlation between cell-associated hemolysis and hemolysis caused by secreted factors was detected. It seems that the cytotoxicity of the studied P. aeruginosa strains was determined by ExoU and ExoS third type secretion effectors.

[The experience of implementation of REP-u RAPD-polymerase chain reaction in epidemiologic characteristic of nosocomial isolates Pseudomonas aeruginosa].

The article presents comparative evaluation of diagnostic value of technique REP- u RAPD-polymerase chain reaction applied under genetic typing of clinical isolates of Pseudomonas Aeruginosa. The strains are isolated in different hospital departments of medical institutions in adult (8 medical institutions; n = 145) and children (5 medical institutions; n = 151) medical networks. The results of study demonstrated different boundary capacity of three reactions. The Simpson discrimination index made up to 0.993, 0.875 and 0.639 for RAPD-, ERIC- and BOX-polymerase chain reaction correspondingly. The RAPD-polymerase chain reaction makes it possible to detect individual characteristics of strains. Out of two alternatives the REP-polymerase chain reaction demonstrated its advantage, besides only with one primer ERIC2. The BOX-polymerase chain reaction has a least discriminating capacity under typing of isolates P. aeruginosa, detecting only species' characteristics. The clinical strains P. aeruginosa are distributed on 24 genome groups and 52 isolates had individual genotypes. The evaluation of results of genetic typing permitted to point out both similarity of tendencies in propagation of strains of P. aeruginosa among hospitalized adults and adolescents and specificity of detection in neonatal clinics. It is obvious that hospitals of different profiles, including departments of reanimation and intensive therapy represent specific ecological environment significantly different in its level of endogenous and exogenous infection.

[Prevalence of cytotoxicity effectors in nosocomial Pseudomonas Aeruginosa strains].

AIM: Analysis of occurrence of the third type secretory system (TTSS) effectors in clinical P. aeruginosa strains. MATERIALS AND METHODS: Intra-hospital (n = 164) and extra-hospital (n = 30) strains of P. aeruginosa were studied. Detection of exoS and exoU genes was carried out by PCR in DNA Engine Dyad Thermal Cycler ("Bio-Rad", USA). Metallo-beta-lactamase (MBL) producers were detected by the presence of blaVIM-2 gene. RESULTS: Screening of intra- and extra-hospital strains for the presence of genes coding ExoS and ExoU showed, that exoS is detected in genome of clinical isolates in 59.8% and exoU--31.1% of cases. At the same time, strains with exoS-/exoU+ genotype predominated in lCU (Φ = 0.466; p = 0.0000). A significant association between the presence of the respective effectors and material of strain isolation was not detected. exoU gene was more frequently detected in genome of MBL producers (Φ = 0.784; p = 0.0004). CONCLUSION: A significant association between exoU and blaVIM-2 could be explained by clonal prevalence of P. aeruginosa ST235 VIM-2, circulation of those is noted on all the territory of Russia. As a rule, ExoU is produced by highly virulent poly-antibiotic resistant hospital isolates that determine unfavorable outcomes of pseudomonas infection.

[The experience of applying techniques of molecular genetics in identification of clinical strains Pseudomonas aeruginosa].

The article presents comparative analysis of application of common bacteriologic and molecular techniques to identify P. aerugenosa. The genetic detection was applied using polymerase chain reaction with genus-specific and species-specific primers and amplification of conservative sites of gens 16S pRNA with successive identification of bacteria by sequenation. It is established that 95% (151) of strains correspond to species of P. aerugenosa detected in primary bacteriologic laboratories and only 8 strains were not blue pus bacillus. Most of strains were closely congenial species: 2 isolates belonged to Pseudomonas aeruginosa group, 3 isolates to Pseudomonas putida group, 2 strains to Comamonas species and 1 isolate to Stenotrophomonas maltophilia species. The effectiveness of cultural technique of laboratory diagnostic was demonstrated concerning infections conditioned by P. aerugenosa. This conclusion does not eliminate application of molecular genetic trechnologies in complicated arbitral cases of bacteriologic analysis during monitoring of nosocomial infections.

[Effect of Pseudomonas aeruginosa exometabolites on planktonic and biofilm cultures of Escherichia coli].

AIM: Study the effect of P. aeruginosa exometabolites on planktonic and biofilm cultures of bioluminescent E. coli strain. MATERIALS AND METHODS: E. coli K12 TG1 (pF1 lux+ Ap(r)) recombinant bioluminescent strain, P. aeruginosa ATCC 27853 reference strain and 2 nosocomial isolates were used. Pyocyanin and pyoverdin content in supernatant of P. aeruginosa over-night cultures was evaluated according to E. Deziel et al. (2001). Planktonic and biofilm cultures of E. coli were obtained in 96-well plates (LB, statically, 37 degrees C), optical density of plankton, film biomass (OD600, OD580) and bioluminescence in plankton and biofilm were evaluated in microplate reader Infiniti M200 (Tecan, Austria). RESULTS: P. aeruginosa exometabolites increased the duration of lag-phase in E. coli, and short term exposition inhibited luminescence of planktonic cells. These effects are determined by bactericidal action ofpyocyanin and pyoverdin. Supernatants ofover-night cultures of P. aeruginosa inhibit formation of biofilm and disrupt the formed biofilm of E. coli. Effect of pyocyanin and pyoverdin on these processes is not established, other factors may have higher significance. CONCLUSION: Bioluminescence of E. coli K12 TGI that reflects the energetic status of the cell allows to evaluate and prognose the character of coexistence of P. aeruginosa in combined with E. coli planktonic and biofilm culture.

[Formation of biofilms bynosocomial Pseudomonas aeruginosa strains].

AIM: Analysis of biofilm forming ability of nosocomial Pseudomonas aeruginosa strains. MATERIALS AND METHODS: 148 P. aeruginosa strains were used in the study. Reference strain P. aeruginosa 27853 was used as control. Biofilm forming was studied on the surface of96-well polystyrol plate. RESULTS: Most of the P.aeruginosa strains isolated both in intensive care (75.5%) and in other units (76.8%) of all the health care institutions were forming films, but ICU group displayed high film forming activity more frequently. The highest indexes of biofilm forming ability were detected in strains isolated from washouts of catheters, sputum and wound discharge. A high correlation between film forming and haemolytic activity for cultures isolated from wound discharge (r = 0.852, p < 0.05) and sputum (r = 0.816, p < 0.05) was detected.IIn the latte rgroup a correlation between film forming and phospholipase activity (r = 0.677, p < 0.05) was also observed. Film forming process was more intensive at 20 degrees C in all the groups. In most of the cases intensity of film forming by P. aeruginosa was independent of the presence of metabolites and of other bacterial taxa. CONCLUSION: High film forming activity provides competitiveness of strains, and probably can be one of the markers of nosocomial isolates.

[Diagnosis of chlamydial infections in man and animals]. / Diagnostika khlamidiinykh infektsii cheloveka i zhivotnykh.

In this article the problems of immunodiagnostics of generalized and local chlamydial infections are discussed. Extensive material is used for the substantiation of the expediency of using serological methods for diagnosing zoonotic and anthroponotic chlamydial infections. A rational scheme of using the passive hemagglutination (PHA) test and the complement fixation test for the detection of generalized chlamydial infections, as well as the PHA test and the indirect immunofluorescence test for diagnosing urogenital chlamydial infections.

[Increasing the effectiveness of the therapeutic action of probiotics]. / Povyshenie éffektivnosti terapevticheskogo deistviia probiotikov.

The problems of the optimization of the therapeutic action of probiotics are discussed. Their use in combination with vitamin-enriched food additives is recommended. Balsams "Beryozka" and "Zolotoi koren" have been found to promote the adhesive capacity of bifidobacteria and lactobacteria. At the same time "Zolotoi koren" also enhances the functional activity of lactobacilli. The increase of the adhesive capacity and metabolic activity of automicroflora on one hand and the pronounced biostimulating effect of the balsams on the other hand may ensure a sufficiently high level of the colonization resistance of the body.