[Effect of Fitofron on the persistence factors of opportunistic pathogens in urine isolated from patients with urinary tract infections].

AIM: To perform an experimental evaluation of the effect of Phytofron, used for the treatment of urinary tract infections, on the ability of opportunistic pathogens to inactivate innate immunity factors (lysozyme, pro- and anti-inflammatory cytokines) and form biofilms. MATERIALS AND METHODS: In vitro experiments were carried out on clinical isolates from urine of patients with pyelonephritis and cystitis: Escherichia coli, Staphylococcus aureus, S. haemolyticus, S. epidermidis, Enterococcus faecalis. The effect of Fitofron NPO FarmVILAR (Russia) on the anticytokine activity of bacteria against regulatory cytokines (IL4, IL6, IL8, TNF and IL17A) was determined by enzyme immunoassay, while anti-lysozyme trait and the ability to form biofilms was evaluated by the photometric method. RESULTS: The inhibitory effect of Fitofron on the ability of opportunistic microorganisms to inactivate innate immunity factors (lysozyme) and form biofilms, as well as the predominant inhibition of the studied cytokines, was experimentally established. CONCLUSION: Inhibition of the persistence factors of opportunistic pathogens by Fitofron, documented in vitro, can be considered as one of the possible mechanisms of its biological activity in vivo.

[Effect of Prostanorm on the persistent potential of microorganisms isolated from patients with chronic bacterial prostatitis].

AIM: To determine the effect of the Prostanorm on the persistent potential of microorganisms isolated from the prostatic secretion of patients with chronic bacterial prostatitis. MATERIALS AND METHODS: In vitro experiments were carried out on uropathogens, isolated from prostate secretions in patients with chronic bacterial prostatitis, including Escherichia coli, Staphylococcus aureus, S. haemolyticus, S. epidermidis, Enterococcus faecalis. The effect of Prostanorm (liquid extract for oral use) of NPO FarmVILAR (Russia) on the anti-cytokine activity of bacteria against regulatory cytokines (IL4, IL6, IL8, TNF and IL17A) was evaluated by enzyme immunoassay, while anti-lysozyme trait and the ability to form biofilms was evaluated by the photometric method. RESULTS: Inhibitory effect of Prostanorm on the anti-lysocyme activity and the ability of microorganisms to form biofilms was found. A decrease in the anticytokine activity against IL17A and TNFa in all studied species of microorganisms was seen, including anticytokine activity against IL4, IL6, IL8 in Escherichia coli and against IL6 and IL8 in coagulase-negative staphylococci. CONCLUSION: The ability of Prostanorm to reduce the persistence factors of opportunistic microorganisms in vitro is one of the possible mechanisms, proving its prostatotropic action, bacteriostatic and anti-inflammatory activity.

[Antibiotic resistance of strains of gram-positive cocci isolated from prostate secretion in men with chronic bacterial prostatitis].

OBJECTIVE: to characterize the antibiotic resistance of gram-positive cocci strains isolated from the prostate secretion in men with chronic bacterial prostatitis at the level of phenotype and genotype. MATERIALS AND METHODS: Bacteria were isolated from the prostate secretion of men of reproductive age (20-45 years) with chronic bacterial prostatitis by conventional bacteriological method. The type of microorganisms was determined by MALDI-TOF mass spectrometry. Resistance to 16 antibiotics of 31 strains of E. faecalis and 91 cultures of coagulase-negative staphylococci (CoNS) was determined by the disk diffusion method. Antibiotic resistance genes (mecA; blaZ; aac(6)- aph (2); ant (4)-Ia; aph (3)-IIIa; gyrA, grlA) were detected using polymerase chain reaction (PCR ) using selected primers. RESULTS: A high resistance of enterococci to antibacterial drugs was revealed: fluoroquinolones, carbapenens, cephalosporins (with the exception of cefoperazone), gentamicin and oxacillin. It has been established that CoNS are characterized by variable antibiotic resistance, while: isolates of S. epidermidis and S. haemolyticus are resistant to all studied fluoroquinolones and carbapenems; S. warneri to carbapenems and the vast majority of studied cephalosporins; S. saprophyticus - to aminoglycosides. Amoxiclav and cefoperazone are characterized by the highest activity against clinical isolates. Using PCR, the presence of genetic determinants of resistance to aminoglycosides and -lactams was established in the isolates, with a predominance of the studied genes in CoNS. CONCLUSION: For effective antibiotic therapy in chronic bacterial prostatitis, it is necessary to conduct regional monitoring of the resistance of microorganisms to antibacterial drugs.

Genome Analysis of the Probiotic Strain Enterococcus faecium Ef79OSAU.

We report here the draft genome sequence of Enterococcus faecium strain Ef79OSAU, which was isolated from swine feces. The characteristics of strain Ef79OSAU reveal the absence of pathogenicity factors, a wide range of antimicrobial activity in vitro, and antilisteriosis activity in vivo. Analysis of the E. faecium Ef79OSAU genome revealed a cluster of genes encoding enterocin A without genetic determinants of pathogenicity.

Genomic Sequence of the Strain Enterococcus faecium ICIS 18.

We report here the draft genome sequence of Enterococcus faecium strain ICIS 18, which was isolated from human feces. Analysis of the E. faecium ICIS 18 genome revealed genes encoding resistance to metals, fluoroquinolones, and beta-lactam antibiotics.

[Biological properties of microorganisms isolated from the urine of patients with urolithiasis].

AIM: To define persistent properties and antibiotic resistance of microorganisms isolated from the urine of adult patient undergoing surgery for urolithiasis. MATERIALS AND METHODS: Urine specimens were obtained from the renal pelvis and urinary bladder during percutaneous nephrolithotripsy. Microorganisms that were isolated from the urine were examined for their persistent properties (anti-lysozyme activity, the ability of biofilm formation) and antibiotic resistance using photometric and bacteriological methods. RESULTS: Strains of microorganisms isolated from the urine of patients with urolithiasis have high anti-lysozyme activity and the ability of biofilm formation, and variable antibiotic resistance. These properties should be taken into account when selecting an empirical antibiotic therapy for preventing infectious-inflammatory complications after percutaneous nephrolithotripsy. CONCLUSION: The high level of resistance of microorganisms isolated from the urine of patients with urolithiasis to the studied antibiotics, their ability to inactivate lysozyme and form biofilms may be the cause of the development of postoperative complications.

Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58.

Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin-derived novel synthetic peptide In-58. In-58 was generated by replacing all tryptophan residues on phenylalanine in D-configuration; the α-amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In-58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In-58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD'::lux), we investigated the action of indolicidin and In-58 at the subcellular level. At subinhibitory concentrations, indolicidin and In-58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

[Structure of microorganism species cultured from urine of urolithiasis patients].

AIM: To investigate the incidence of microorganisms of different taxonomic groups and their associations in the pelvic and bladder urine of adult urolithiasis patients. MATERIALS AND METHODS: A bacteriological method and metagenomic sequencing were used to investigate the bacterial spectrum of microflora cultured from pelvic and bladder urine sampled during surgical interventions in urolithiasis patients. RESULTS: The both microbiotas had approximately the same spectrum, but in 26.1% of patients it was inconsistent. Metagenomic analysis detected DNA of microorganisms in urine samples which were found free of microflora by the bacteriological method. CONCLUSION: The study findings showed species diversity of microorganisms cultured from pelvic and bladder urine sampled during surgical interventions in urolithiasis patients.

Novel haemoglobin-derived antimicrobial peptides from chicken (Gallus gallus) blood: purification, structural aspects and biological activity.

AIM: To purify and characterize antimicrobial peptides derived from the acid extract of Gallus gallus blood cells. METHODS AND RESULTS: Two polypeptides (i.e. CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (G. gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the purified peptides were similar and were 4824·4 and 4825·2 Da, which have been measured by matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS). Their amino acid sequences were determined by Edman degradation and showed that the peptides were fully identical to the two fragments of G. gallus α-haemoglobin localized into different subunits (A and D respectively). The peptides were active in micromolar concentrations against Gram-negative Escherichia coli K12 TG1. Using the 1-N-phenylnaphthylamine, the FITC-dextran labelled probes and the live/dead staining allowed to show the hemocidin mode of action and estimate the pore size. CONCLUSION: In this study, for the first time, α-haemoglobin from chicken (G. gallus) has been investigated as a donor of the two high homologous native peptide fragments that possess potent antibacterial activity in vitro. These are membrane-active peptides and their mechanism of action against E. coli involves a toroidal pore formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results expand the perception of the role of haemoglobin in a living system, describing it as a source of multifunction substances. Additionally, the data presented in this paper may contribute to the development of new, cost-effective, antimicrobial agents.

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES FROM CHICKENS THROMBOCYTES].

AIM: Isolation and study of biological activity of antimicrobial peptides from chickens thrombocytes. MATERIALS AND METHODS: Peptides from chickens thrombocytes, obtained by reverse-phase high-performance liquid chromatography method with stepped and linear gradients of concentration increase of the organic solvent were used in the study. Their antimicrobial activity was determined by microtitration method in broth; mechanism of biological effect--by using fluorescent spectroscopy method with DNA-tropic dyes. RESULTS: Individual fractions of peptides were isolated from chickens thrombocytes, that possess antimicrobial activity against Staphylococcus aureus P209 and Escherichia coli K12. A disruption of integrity of barrier structures of microorganisms under the effect of thrombocyte antimicrobial peptides and predominance of cells with damaged membrane in the population of E. coli was established. CONCLUSION: The data obtained on antimicrobial activity and mechanism of bactericidal effect of the peptide fractions from chickens thrombocytes isolated for the first time expand the understanding of functional properties of chickens thrombocytes and open a perspective for their further study with the aim of use as antimicrobial means.

[Antibiotic Resistance of Enterococci Isolated from Healthy Humans and Patients with Various Pathologies.]

High resistance of enterococci to the currently used antibacterials, such as tetracycline, ciprofloxacin and ampicillin was observed. Streptomycin, gentamicin and vancomycin showed the highest activity against the clinical isolates of Enterococcus spp. Streptomycin and gentamicin showed the highest activity against the intestinal enterococci. The PCR revealed the presence of the genetic determinants of resistance to aminoglycosides, glycopeptides and tetracycline in the isolates. The comparison of the results of the bacteriological and genetic tests provided detection of fecal and clinical isolates of Enterococcus spp. possessing the genes of resistance to aminoglycosides and glycopeptides, still without the finally developed significant clinical resistance to the above antibacterials.

[BIOLOGICAL PROPERTIES OF ENTEROCOCCI OF VARIOUS ORIGINS].

AIM: Study the prevalence and intensity of anti-hemoglobin and anti-lactoferrin activity in enterococci as representatives of symbiotic intestine microflora and causative agents of infection-inflammatory diseases in children. MATERIALS AND METHODS: 95 strains of enterococci were used in the study, that were isolated from intestines of children during examination for dysbiosis, and 34 clinical isolates. Strain identification was carried out by using multiplex PCR. Anti-lactoferrin and anti-hemoglobin activity were determined by O.V. Bukharin et al. (2005). RESULTS: A higher level of prevalence and intensity of anti-lactoferrin, anti-hemoglobin activity, as well as a combination of those properties was established in clinical isolates of Enterococcus faecaIis and E. faecium compared with strains, isolated from intestines of healthy individuals. CONCLUSION: The studied properties could be used for differentiation of pathogenic and non-pathogenic strains of enterococci and during selection of inhibitory preparations, suitable for therapy of enterococci infections.

[PHENOTYPICAL CHARACTERISTICS AND GENETICAL DETERMINANTS OF PATHOGENICITY OF STAPHYLOCOCCUS AUREUS, ISOLATED FROM BACTERIAL CARRIERS, RESIDING ON THE TERRITORIES WITH VARIOUS LEVELS OF ANTHROPOGENIC POLLUTION OF AIR ENVIRONMENT].

AIM: Comparative phenotypical and genetical evaluation of pathogenic potential of Staphylococcus aureus strains, isolated from resident bacterial carriers, residing on the territories with anthropogenic pollution of air environment of varying intensity. MATERIALS AND METHODS: S. aureus, isolated 3 times from mucous membrane of the anterior of nose from 210 children, were the object of the study. Anti-carnosine activity and biofilm formation was determined by a photometric method, antibiotics resistance--by a disc diffusion method. lukS, lukF, sec 3, clfA, clfB, agr and mecA gene detection, that are associated with S. aureus, was carried out by PCR. RESULTS: S. aureus strains, isolated from children, residing on the territories with a high level of anthropogenic pollution of air environment, were characterized by antibiotics resistance, higher values of anti-carnosine activity, 2 times more frequently formed biofilms with higher values of the parameter. clfA and clfB genes, that determine colonization of mucous membranes, and agr gene were detected in all the studied S. aureus strains, lukF and sec 3 genes were detected in 20-40% of the strains, isolated from children, residing on both territories. mecA and lukS genetical determinants were not detected. CONCLUSION: S. aureus, isolated from children, residing on the territories with high levels of anthropogenic pollution of air environment; were characterized by higher values of the studied factors of persistence and stability against antibiotics. Genetical determinants of pathogenicity were not detected in S. aureus, isolated from individuals, residing on both territories.

[Cycloferon biological activity characteristics].

AIM: Study the effect of cycloferon in experimental and clinical conditions on persistence properties of aurococci as well as features of their morpho-functional reaction by atomic force microscopy. MATERIALS AND METHODS: The study was carried out in 12 Staphylococcus aureus clones isolated from mucous membrane of nose anterior part of a resident carrier. The effect of cycloferon in vivo was evaluated in 26 resident staphylococci carriers under the control of anti-carnosine activity of staphylococci. Anti-carnosine activity was determined by O.V. Bukharin et al. (1999), biofilm formation -by G.A. O'Toole et al. (2000). Staphylococci treated with cycloferon were studied by atomic force microscopy in contact mode using scanning probe SMM-2000 microscope. RESULTS: The decrease of persistence properties of staphylococci under the effect of cycloferon in vitro and in vivo may be examined as one of the mechanisms of biological activity of the preparation. A significant increase of S. aureus surface roughness and changes in their morphology under the effect of cycloferon allow stating the disorder of barrier functions in the aurococci cell wall. CONCLUSION: The data obtained expand the understanding of cycloferon biological activity mechanisms.

[Characteristic of microflora isolated during mechanical lung ventilation].

AIM: Study species composition and biological properties of microorganisms isolated from lower respiratory tract of 34 patients of surgical departments during mechanical lung ventilation. MATERIALS AND METHODS: 59 strains of microorganisms were isolated and identified by established methods from tracheobronchial secretion during intubation and extubation. Hemolytic and anti-lysozyme activity was determined in isolates by methods described previously (O.V. Bukharin, 1999). RESULTS: During extubation an increase of seeding of tracheobronchial secretion in patients, increase of microorganism species diversity and frequency of occurrence of highly virulent strains characterized by the ability to hemolyze erythrocytes and high values of anti-lysozyme activity were established. CONCLUSION: The data obtained may be used to prognose the risk of development of diseases of respiratory system.

[Characteristic of virulence potential of clinical isolates of enterococci].

AIM: Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. MATERIALS AND METHODS: 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic--by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylL1, ESP, HYL, ASA)--by using PCR. RESULTS: Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. CONCLUSION: Genetic determinants of virulence are more widespread among clinical isolates of E.faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.

[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.

[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.

[Features of microbiocenosis of the large intestine in dysbiotic disorders].

AIM: To determine features of intestinal microbiocenosis in dysbiosis as well as biological characteristics of isolated microflora in residents of Orenburg city. MATERIALS AND METHODS: 70 children one year old and 60 adult 1 - 60 years old were examined for dysbiosis. Bacteriologic identification of the large intestine's content was performed using method of serial dilutions. Isolated microorganisms were identified by routine methods. Assessment of the degree of dysbiotic disorder was conducted according to the standard guideline "Patients management protocol. Intestinal dysbiosis". Antibiotic resistance of Klebsiella species was determined by disc-diffusion method, antagonistic activity of lactobacilli--by plate culture method, and lysozyme activity--by agar bullet method. RESULTS: Dysbiotic disorders were registered in more than 90% of examined subjects. For patients of both age groups, stage I of intestinal dysbiosis was observed most often. Dysbiotic disorders were characterized by increased amount of bacteria from Klebsiella genus and yeast-like fungi from Candida genus. It was established that antibiotic resistance was widely prevalent in isolated strains of Klebsiella. At the same time representatives of normal microflora, i.e. lactobacilli, had a marked antagonistic activity against Klebsiella species, Staphylococcus aureus, Candida fungi and low level of lysozyme activity. CONCLUSION: Among the population of Orenburg, intestinal dysbiosis was widely prevalent and characterized by predominance of Klebsiella spp. and Candida spp. among opportunistic microflora. One of the rational methods of correction of compensated forms of intestinal dysbiosis is stimulation of growth of normal flora including lactobacilli, which have antagonistic activity.

[Structural and functional characteristics of betulin derivatives].

The effect of betulin derivatives on persistence properties of microorganisms was studied in vitro. It was shown that the antipersistence action of the betulin derivatives depended on their structure and the microbial species. The experimental data on the structure - function relation could be useful in development and synthesis of new agents for therapy of chronic infections associated with persistence of bacterial pathogens in macroorganism.