Vol 89, No 1 (2012)

Aim. Study of taxonomical structure of wound infection agents, prevalence of mixes, and detection of character of their possible connection with the results of various microorganisms population interaction in septic wounds. Materials and methods. A microbiological study of material from patients with wound infection (WI), 582 of those were cured in reanimation and intensive therapy departments (RITD; group 1) and 1455 - in surgical departments (SD; group 2), was performed. Taxonomic membership and ability to coexist was determined in 4129 microorganisms strains. Etiological role of the agents was evaluated by using values of consistency rate (CR). Species that were present in more than 50% of samples were considered consistent, in 25 to 50% - additional, and in less than 25% - random. Frequency rates (FR) were also determined, that is the fraction of a certain species (genus) of the microorganism (in %) from all the isolated cultures that correspond to 100%. For the determination of the significance of individual species of the agent in the structure of mixed microorganism populations, FR - their fraction (%) in mixed population from the number of strains of this species that correspond to 100% - was calculated. Results. A significant part of the microorganisms strains, more frequently in reanimation department (65.5%), caused wound suppuration in populations mixed with other species of the agents. In reanimation and surgical departments consistent species of wound infection agents were not detected. A leading etiological role of Staphylococcus aureus (FR 19.2% and 23.9%) was determined, and FR of S. aureus strains in mixes was 64.6% in RITD and 46.8% in SD. The parameters of other agents of WI in the comparison groups were similar. However FR among mixes in RITD were sig- nificantly higher for streptococci that do not belong to S. pyogenes species (72,5%), and also nonfermentative microorganisms (67,2%), and in SD - in Klebsiella pneumoniae mixes. For agents of wound infection especially in RITD, low species diversity was characteristic and the number of mixes variants is significantly higher. In RITD mixed infections develop more frequently, and the ecological community of microorganisms reaches higher values than in SD. Conclusion. During the analysis of microbiological data in RITD and SD general patterns and specific features of taxonomical structure, prevalence of mixed populations and character of their ecological community in wound infection was determined.

Aim. Complex characteristic by phenotype signs and main virulence genes of Yersinia enterocolitica strains circulating in various regions of Russian Federation. Materials and methods. 46 strains of Y. enterocolitica of 2 - 4 biotypes and 401 strains of Y. enterocolitica 1A biotype isolated in 15 administrative territories of Russian Federation (Siberian, Far Eastern, Northwestern, Urals Federal Districts) from infected people, rodents, agricultural animals, birds, the environment were studied. Phagotyping was performed in the reference laboratory of the Pasteur Institute (Paris). All the Y. enterocolitica cultures were studied for the presence of ail, ystB and ystA genes by PCR method. Presence of virulence plasmid pYV was determined by gel electrophoresis by T. Kieser method. Results. 447 strains of Y. enterocolitica biotype 1A and 2 - 4 were studied. Most of the strains belonged to serotypes О:3; О:9; О: 5; О: 6,30; О:6,31; О:7,8. Phagotyping was performed for part of the strains. Phagotypes Xz and Xo were determined in biotype 1A strains. 2 - 4 biotype strains circulating in Siberia and the Far East were characterized by phagotype VIII, X3 that are present in other countries, and phagotype Xz that is spread only in Russia. Phagotypes IXa, IXb, II that are characteristic for strains from Canada, South Africa, Japan were not detected in Russian Federation. All the strains of 2 - 4 biotypes had ail and ystA genes. Most of the recently isolated strains had pYV. The only pathogenicity factor detected in 81.3% of biotype 1A strains including 14 strains from patients was ystB gene. These infections were accompanied by an expressed clinical symptomatology of enteritis and enterocolitis. Conclusion. Isolation of 1A biotype strains from patients necessitates execution of diagnostic studies of intestinal yersiniosis in patients with diagnosis «acute intestinal infection of undetermined etiology».

Aim. Study dynamics of IFNƒ, IFNƒ, TNFƒ cytokines in healthy adults after administration of inactivated subunit monovalent influenza vaccine, А/California/7/2009 (H1N1) strain. Materials and methods. Levels of IFNƒ, IFNƒ, TNFƒ cytokines were studied in blood sera of 58 mostly healthy adults aged 18 - 60 years. Kits for enzyme immunoassay determination of cytokine levels (Vector-Best, Novosibirsk) were used in the study. Antibody titers to А/California/7/2009 (H1N1) strain were determined at analogous time by using microneutralization reaction (MNR). Results. Changes in the level of IFNƒ, IFNƒ, TNFƒ in healthy volunteers immunized by pandemic influenza vaccine were evaluated. Vaccine was safe. Two immunizations did not result in an increase of TNFƒ level that is an additional evidence of vaccine safety. IFNƒ level had a tendency to increase in vaccinated volunteers. IFNƒ levels in volunteers with normal level of this cytokine (below 10 pg/ml) were increased significantly after the second immunization (from 2.66±2.48 to 5.21±2.56). Correlation analysis showed that there is a strong negative association between IFNƒ, IFNƒ and seroconversion.

Aim. Study of antagonistic activity of lactobacilli of the colon against members of its autochthonous bacterial flora and agents of some acute infectious and chronic diseases of the gastrointestinal tract. Materials and methods. Antagonistic activity of 19 lactobacilli cultures against 28 cultures of bacteria belonging to various groups and fungi was evaluated within the framework of specially developed two-stage cultivation technique in the conditions of a combined system. The results of the study were evaluated according to a semi-quantitative scale that allows to put one or the other value of the zone of growth delay of the studied strain culture in compliance with the one or the other (low, moderate, high) level of antagonistic activity of the lactobacillus culture. Results. Lactobacilli of the colon showed selective antagonistic activity against pathogenic enterobacteriae: pronounced against Salmonella enterica Serovar Enteritidis, Shigella flexneri 2b, Yersinia spp., and trace against Salmonella enterica Serovar Typhimurium. The level of antagonistic activity of lactobacilli against a wide range of members of autochthonous bacterial flora varied in a wide range, without revealing connection neither to its belonging to species, nor to its population level, nor to the belonging to group of the antagonistic effect objects. On the other hand a connection was traced with belonging to a certain microbiota: being quite active against members of its own microbiota, lactobacilli often showed significantly lower level of antagonistic activity against cultures with the same species name isolated from other microbiota. Conclusion. In light of the results obtained, level of lactobacilli population may hardly be viewed as the only criteria of their full participation in the process of stabilizing microecological welfare of the colon, that allows to make a complete representation of the level of dysbiotic disorder in the mentioned biotope. With in the framework of rational bacteriological diagnostics of the level of dysbiotic disorders in the colon, evaluation of population level of lactobacilli should be evaluated along with the degree of their antagonistic activity against other components of the same microbiota.

Aim. Isolation and composition comparison of extracellular antigens (ECA) of pathogenic burkholderiae in SDS-PAGE electrophoresis and their use for differentiation of these microorganisms by immunodiffusion methods. Materials and methods. 60 Burkholderia pseudomallei strains, 14 B. mallei strains, 5 B. thailandensis strains, 4 B. cepacia strains were studied. ECA was obtained by Liu technique on F-agar covered with cellophane. SDS-PAGE electrophoresis was performed in 10% gel by Laemmli, immunodiffusion reaction (IDR) in 1% agarose gel, IDR with live cultures, immunoelectrophoresis (IEPH) was performed by the standard techniques. Sera was obtained by immunizing rabbits with a mixture of ECA and incomplete Freund adjuvant. Results. ECA spectra of typical strains of the studied burkholderiae strains after the electrophoresis in SDSPAGE stained by silver have 8 - 9 major fractions. ECA electrophoregrams of B. pseudo - mallei and B. thailandensis had a high simi larity. ECA analysis by IDR with antisera against ECA revealed maximum number of cross-reactive ECA (3) between B. pseudomallei и B. thailandensis. These strains had only a single crossreactive ECA to B. mallei strain. IDR with live culture and antisera to B. thailandensis ECA revealed ECA in all the B. pseudomallei, B. thailandensis strains and did not reveal those in B. mallei strains. Analysis of electrophoregram obtained with IEPH method of pathogenic burkholderiae ECA with antisera to ECA revealed differences of the composition sufficient for their differentiation. Conclusion. The differences of ECA composition revealed by immunodiffusion methods allowed to develop additional approaches of differentiation of glanders and melioidosis pathogenic agents.

Цель. Изучение гетерогенности вируса гепатита В у взрослых пациентов с хроническим гепатитом В и определение диагностических возможностей современных тест-систем при выявлении HBsAg с аминокислотными заменами в области главного гидрофильного региона (MHR). Материалы и методы. В 27 изолятах вируса гепатита В, выделенных у пациентов с хронической инфекцией вирусом гепатита В, проживающих в г. Владимире, определена нуклеотидная последовательность участка генома, соответствующего preS1/preS2/S генам. Результаты. Во всех 27 изолятах обнаружен вирус генотипа D, представленный тремя субгенотипами - D1, D2 и D3, выявленными в 18%, 26% и 56%, соответственно. Исходя из распределения нуклеотидных замен в сравниваемых функциональных участках генома ВГВ: сайт, кодирующий вход вируса в клетку (2875 - 2991 н.о.), pre-S2/S промоторный участок (2994 - 3171 н.о.), 5'-концевые последовательности pre-S2 и S-генов (3172 - 154 и 155 - 455 н.о.), MHR (455 - 635 н.о.) и 3'-концевая последовательность S-гена (636-835 н.о.) установлено, что замены сконцентрированы главным образом в промоторном участке S2/S-генов (30,8%). По предсказанной аминокислотной последовательности в 24 из 27 образцов были определены серотипы HBsAg, причем в 17 случаях HBsAg относился к серотипу ayw2 (71%) и в 7 случаях - к серотипу ayw3 (29%). В 5 изолятах идентифицированы аминокислотные замены G145A, M133I, S132T, локализованные в главном гидрофильном регионе, и P217L, S207N, V184A, локализованные в С-конце белка S, связанные с диагностическим и вакцинным ускользанием. Заключение. Изучены диагностические возможности тест-систем при определении HBsAg с известной аминокислотной последовательностью в области MHR. Показаны приблизительно одинаковые возможности шести тест-систем выявлять HBsAg с наличием аминокислотных замен G145A, M133I и S132T, локализованных в MHR.

Aim. Study of innate immunity genes expression (TLR9 and BD-2) in epithelial cells of mice cornea on the model of viral keratitis during the application of immunotherapy. Materials and methods. RNA isolation from cornea cells was performed by using RNeasy Mini Kit (Qiagen) and «RIBO-sorb» (ILS, Russia) reagent kits. «Kit for performing PCR-RT in the presence of intercalating dye SYBR Green I» (Syntol, Russia) was used for performing PCR. Data on gene expression is presented in decimal logarithms (relatively to 1 million of ƒ-actin gene copies). Results. In C57Bl/6 mice line at day 1 post infection the virus was detected in 33% of animals, at 3 days - in 90%, at 7 days the number of infected mice started to decline. In cornea of the mice infected with HSV-1 a significant decrease of TLR9 gene expression was observed. Administration of a complex of natural cytokines and antimicrobial peptides (Superlimf preparation) caused statistically significant increase of expression levels of the studied TLR9 and BD- 2 genes. Conclusion. The model of viral keratitis and expression analysis of recognizing receptor TLR9 and defensin (BD-2) genes can be used to study mechanism of action of various preparations during infection in eye tissues and evaluation of possibility of their use in complex therapy of ophthalmic herpes.

Aim. Study parameters of oxidative stress and apoptosis of neutrophils in patients with ankylosing spondylitis, systemic lupus erythematosus, systemic scleroderma and rheumatoid heart disease. Materials and methods. 240 patients with rheumatoid diseases and 25 healthy control group volunteers were examined. Neutrophil isolation from peripheral blood was performed by using double density gradient of ficoll-urografin. Cell functional activity was studied by chemiluminescence method. Pro-apoptosis antigen bak expression by neutrophils was studied by using streptavidin-biotin method. Griss reagent was used for nitrogen oxide production analysis. Results. An increase of oxygen dependent neutrophil metabolism processes was detected in patients with ankylosing spondylitis in comparison with systemic lupus erythematosus and systemic scleroderma patients. Systemic lupus erythematosus is characterized by higher biocidity of neutrophils in comparison with systemic scleroderma and rheumatoid heart disease. Increase of neutrophil granulocyte activity in ankylosing spondylitis is accompanied by an increase of superoxide-anion formation. In spite of high level of metabolic activity neutrophils in patients with ankylosing spondylitis have a low functional reserve, and neutrophils in patients with systemic scleroderma have the highest reserve potential. Conclusion. Differences in expression parameters of oxidative stress by neutrophils depend on nosological form, varying by production level and active oxygen form formation reserve.

Features of hantavirus pulmonary syndrome are considered in the review - zoonosis natural focal polyetiological viral infection, that is characterized by lung injury. Etiology of the disease, main characteristics of the agents, epidemiology, contagiousness, pathogenesis, clinical presenta - tion of this pathology are examined. Laboratory diagnostics, therapy and prophylaxis of hantavirus pulmonary syndrome are described.