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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.

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AIM: Determine prevalence of genetic determinants of virulence among enterococci strains comprising human intestine microbiota. MATERIALS AND METHODS: 81 enterococci strains isolated from intestine of individuals during examination for dysbiosis were used in the study. Strain identification was performed by using multiplex PCR. Hemolytic and gelatinase activity was determined by dish method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, esp)--by PCR. RESULTS: A wide set of genetic determinants of virulence was detected in E. faecalis strain microorganisms. CONCLUSION: Enterococcus genus microorganisms of human intestine microbiota that have virulence potential may become the reason for endogenous infection. The data obtained may be used for prognosis of risk of development of endogenous enterococci infections.

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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.

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AIM: Genetic characteristics of Enterococcus faecium strains isolated from human intestine in Russia. MATERIALS AND METHODS: Seven strains of E. faecium with antimicrobial activity against Gram-positive bacteria and yeast fungi were isolated from persons aged 4 months - 44 years. Using multilocus sequence-typing, sequences of internal fragments of genes of general metabolism (adk, atpA, ddl, gyd, gdh, purK, pstS) were determined. RESULTS: Number of alleles for each gene varied from 3 for gdh and pstS to 7 for atpA. Sequence-types of 4 out of 7 cultures of enterococci were described earlier, 3 strains were attributed to new sequence-types. CONCLUSION: Members of identified in this study sequence-types 32, 135, 170, 361 were isolated earlier in other countries from clinical samples (blood, faeces) and hospital environment. Diversity of sequence-types, sources of isolation and significant remoteness of regions where strains belonging to one sequence-type were isolated point to necessity of thorough study of E. faecium evolution.

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AIM: To assess the ability of bacteria and Candida genus' yeast fungi from human fecal microflora to form biofilms. MATERIALS AND METHODS: Thirty-one strains of enterobacteria and 45 strains of yeast fungi from Candida genus isolated from feces of patients during test on intestinal dysbiosis were used. In order to study the ability of microorganisms to form biofilms, level of binding of crystal violet by the formers in sterile 96-well polystyrene plates was studied. RESULTS: Ability to form biofilms was noted in 87.1% of enterobacteria strains. This characteristic was observed in lactose-positive and lactose-negative Escherichia coli and bacteria belonging to Klebsiella, Citrobacter, Serratia, Morganella, Providencia genuses. The highest mean value of the characteristic was noted in strains belonging to Enterobacter cloacae species (6.52+/-2.01). Overwhelming majority of fungal strains belonging to Candida genus formed static biofilms in wells of polystyrene plates. Mean value of the characteristic in group of Candida spp. (not albicans) was significantly higher compared with strains from C. albicans species (2.75+/-0.53 vs. 1.38+/-0.11, p<0.05). The highest value of the mentioned characteristic (6.75+/-1.65) was observed in yeast fungi belonging to C. krusei species. CONCLUSION: Obtained data widen our knowledge about armamentarium of biological characteristics of opportunistic microorganisms (bacteria and fungi) promoting their prolonged persistence in intestinal biotope.

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AIM: To determine features of species composition and factors of persistence of bifidobacteria of human gut microflora in healthy state and during dysbiosis. MATERIALS AND METHODS: Strains of bifidobacteria isolated from feces of patients with gut dysbiosis grades I - III (24 participants) and conditionally healthy persons (14 participants) were used for the study. Isolation and identification of microorganisms were performed by conventional methods. Antilysozyme activity was determined by photometric method and antilactoferrin sign--by immunoenzyme assay. RESULTS: Bifidobacteria isolated from intestinal biotope have differences in species composition, which depended from age and state of microbiocenosis of studied persons. Bifidobacterium bifidum was dominated between strains of bifidobacteria isolated from infants with eubiosis, whereas B. longum dominated in persons > 1 y.o. B. bifidum and B. adolescentis were mainly isolated in infants with gut dysbiosis, where as in children > 1 y.o. B. adolescentis was predominantly isolated. New characteristic of bifidoflora--antilactoferrin activity--was discovered for the first time. Expression of this sign, as well as antilysozyme activity, varied and depended from species of bifidobacteria. CONCLUSION: Obtained data widen our views on species composition of bifidobacteria and discover possible mechanisms of persistence of these microorganisms in intestinal biotope associated with inactivation of host defense factors--lysozyme and lactoferrin.

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The role of anti-lactoferrin activity (ALfA) of bacteria in the persistence phenomenon, i.e. long survival of a causative agent in a host body, was studied in experimental staphylococcal infection. In experimental animals infected with isogenic clones of Staphylococcus aureus with different level of ALfA, longer terms were noted in bacterial recovery of staphylococci with this ability. Analysis on the occurrence and level of ALfA in clinical isolates of bacteria, isolated from different forms of infectious process, confirmed the significance of this sign in the persistence of a causative agent. High values of penetrance and expression of ALfA in bacteria were revealed in strains of microorganisms, isolated from chronic inflammatory diseases and from bacterial carriers.

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The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.

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The detection rate of the antilactoferrin sign and the level of its manifestation in 165 strains of different microbial species, isolated from patients with inflammatory diseases and intestinal dysbacteriosis, were analyzed. The detection rate of antilactoferrin activity was 43 - 90% for Escherichia coli strains, 20 - 86% for Staphylococcus aureus, 60 - 100% for Klebsiella pneumoniae, 76 - 78% for Candida albicans, isolated from different biotopes of man. Most frequently and with high levels of manifestation this sign was registered in strains isolated from the reproductive tract of women. The detection rate of this sign and the level of its manifestation in bacteria of the vaginal and cervical microbiocenosis, isolated from patients, were higher in comparison with healthy persons. The inverse dependence between the level of the antilactoferrin activity of microflora and the content of lactoferrin during the inflammatory process in women was established.

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In the study of 65 microbial cultures isolated from the purulent foci in acute pulmonary abscess and acute pyothorax of 48 patients, a wide spectrum of microflora was detected. Staphylococci and Pseudomonas prevailed among aerobes, bacteroids and peptostreptococci--among anaerobes. In cases of the prolonged course of the pathological process, as compared with the common one, microorganisms exhibited hemolytic activity and high antilysozyme and anticomplementary levels. These findings served as the basis for working out a mathematical model for the prognosis of the disease course with 95% probability.

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The species composition and biological properties of microflora isolated from 42 patients with acute inflammatory pulmonary and pleural diseases were studied. A wide spectrum of aerobic and anaerobic microorganisms was detected. Infective agents causing severe course of the infectious process were found to have a high level of persistence properties.

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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.

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