Vol 92, No 6 (2015)

Aim. Study the spectrum of resistance to antibiotics and its variability of Staphylococcus aureus, Pseudomonas aeruginosa and Вurkholderia cepacia complex (BCC), persisting in lungs of MV patients. Materials and methods. 312 strains of S. aureus, 213 strains of P. аeruginosa, 186 strains of BCC were studied. Monitoring of antibiotics sensitivity was carried out in strains, isolated from 30 patients with chronic S. aureus infection, from 22 patients with chronic BCC infection and from 21 patients with chronic pseudomonas infection. Interval of monitoring was from 14 days to 5 years 7 months. Results. Study of S. aureus, P. aeruginosa and ВCC strains has shown, that 35 and 33.3% of cases of staphylococcus infection, 37 and 46% of pseudomonas infection in children and adults, respectively, 100% of BCC infections were determined by multiresistant clones. Study of genotypically identical strains, isolated from a single patient at different stages, has shown a change in antibiotics sensitivity as a result of persistence. Conclusion. Persistent infection of lungs in patients with MV is determined by exchanging clones with varying antibiotics sensitivity or prolonged circulation of a single clone with a high degree of phenotypical and genotypical variability, that determine alteration of seeding of sensitive and resistant strains from the same patient during monitoring. This confirms the necessity of study of antibiotics sensitivity of strains for prescription of antibacterial therapy.

Aim. Detection of bactericidal effect of pulse-periodic corona discharge (PPCD) on cells and biofilms of Escherichia mli M17. Materials and methods. A gas-discharge device was created based on PPCD in air with power supply parameters: amplitude values of voltage of 30 - 60 kV, pulse repetition rate of 250 - 400 kHz. Ultrastructure changes in cells and biofilms of E. mli M17, affected by PPCD, generated in air, were studied by typical methods of transmission electron microscopy. Results. Disturbances of integrity of surface and abyssal structures of biofilms, as well as changes of morphological properties of E. mli M17 cells, characteristic for sub-lethal heat impact, were detected. Destructive changes of bacterial cells were developed by formation of focal disturbance of cytoplasmic membrane, extension of periplasmic space, formation of globular structures, characteristic for heat effect, and destruction ofcytoplasm. Conclusion. Bactericidal effect ofPPCD on E. mli M17 cells as part ofbiofilms was shown. Destructive morphological changes in cells and biofilms of E. mli M17 after the effect of PPCD were detected for the first time on electron-microscopic level.

Aim. Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira. Materials and methods. 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28°C. Cell extracts were obtained by ethanol/formic acid method. a-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding. Results. Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221,5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classes in Sejroe, Ballum, Tarassovi, Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50% of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae. Conclusion. MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intralaboratory control of leptospira reseeding.

Aim. Monitoring of post-vaccinal complications in children immunized with a parotitis vaccine. Materials and methods. Observation of198 945 children, immunized with 16 lots of parotitis vaccine with Leningrad-3 strain (L-3), was carried out for 3 years. Paired samples of sera and saliva were obtained from children, in whom adverse events were registered for 42 days after vaccination. Titers of specific IgM and IgG were determined in blood sera. Analysis of nucleotide sequences of genes F, SH and NH of RNA of parotitis virus was carried out from samples of blood and saliva. Results. Intensive parameter of vaccine-associated aseptic meningitis under the conditions of the experiments was 0 for 100 000 immunized. Frequency of occurrence of post-vaccinal parotitis was 0.06% from the number of vaccinated - 18 cases of vaccine-associated parotitis were registered and laboratory confirmed. A significant difference in specific activity was detected for 3 lots of the vaccine, that were associated with cases of development of parotitis, relative to that of 13 lots of vaccine, development of parotitis was not registered after administration of those. Conclusion. The study carried out confirmed low neurovirulence of the parotitis vaccine with the L-3 strain of parotitis virus, as well as a low degree of its reactogenicity. A relatively high immunization dose of the used vaccine could be one of the reasons of development of post-vaccinal complications in part of the immunized children.

Aim. Study the effect of osmotic and oxidative stress on survivability and changes in phenotypic and genetic properties of strains of genovariants of V. cholerae El Tor biovar. Materials and methods. 22 strains of V. cholerae El Tor biovar were used in the study. Phenotypic properties of strains were studied in LB medium with the addition of the appropriate ingredients. Surface structures of cells were studied using scanning probe microscope «Solver P47-PRO». PCR was carried out using specific primers in «Tercic» amplificator. Results. After 60 minutes of incubation in 3 M solution of NaCl and after 6 minutes in 20 mM solution of hydrogen peroxide, the amount of surviving cells of genovariants was, respectively, 3.0 - 25.0 and 4.3 - 7.6 times higher than for typical strains. One of the mechanisms of increased resistance of genovariants to high concentrations of salt was associated with the production of an extra exopolysaccharide layer on the cell surface at earlier periods than in typical strains. Osmotic stress results in a reversible reduction of mobility in strains of genovariants of V. cholerae El Tor biovar. Osmotic and oxidative stress was revealed to result in a loss of a number of mobile genetic elements in strains of genovariants. Conclusion. Genovariants of V. cholerae El Tor biovar, that had caused cholera outbreaks in Russia in 1993 - 2001, in contrast to typical strains, isolated in 1970 - 1990, are more resistant to the effect of osmotic and oxidative stress, that, probably, facilitates their higher survivability in both the environment and macroorganism.

Aim. Evaluate role of gene polymorphisms of surfactant proteins in susceptibility and severity of influenza infection course in representatives of Moscow population. Materials and methods. 320 influenza patients, infected with various influenza virus strains, and 115 healthy individuals (control group), were included into the study. Human DNA samples genotyping for determination of SFTPA2 gene rs1965708 and rs1059046, SFTPB gene rs1130866 polymorphisms was carried out using a modified method of «adjacent samples». Results. Most of the individuals of the control group and influenza patients are carries of alleles and genotypes rs1965708 and rs1059046 of SFTPA2 gene, rs1130866 of SFTPB gene, that have, based on scientific literature data, shown association with severe course of influenza А(ШШ) pdm09 and other inflammatory diseases of the respiratory tract. Generally, significant differences in frequency of occurrence of unfavorable genotypes CC rs1965708, АА rs1059046 of SFTPA2 gene and CC rs1130866 of SFTPB gene in influenza patients in comparison with individuals of the control group were not detected, that gives evidence on a high (from 19 to 51%) prevalence of these genotypes in the studied population. Allele C and genotype CC rs1965708 of SFTPA2 gene, allele A and genotype АА rs1059046 of SFTPA2 gene, allele C and genotype CC rs1130866 of SFTPB gene did not shown an association with severe course of А(ШШ) pdm09 influenza. The following pathology registered in most (88%) of the patients with severe course of influenza А(H1N1)pdm09: diseases of cardiovascular (44%), endocrine (36%) and respiratory (12%) systems. Conclusion. Because in most of the deceased patients due to severe course of А(H1Nl)pdm09 influenza, diseases of cardiovascular, respiratory and endocrine system were detected, and an association of unfavorable disease outcome with the studied genetic markers was not detected, dominating risk factor of development of severe course and lethal outcome for А(H1N1)pdm09 influenza in the studied cohort was comorbidity.

Aim. Detection and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). Materials and methods. VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. Results. VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. Conclusion. Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

Aim. Study biological properties of salmonella, isolated from clinical materials and water of Don river. Materials and methods. Salmonella strains of various serovars were used in the study. Biochemical characteristics were studied by generally accepted methods, antigenic properties were evaluated in agglutination reactions, virulence was determined by Dlm for laboratory animals, antibiotics sensitivity was verified by disc-diffusion method. Results. The presence of pathogenicity factors in isolated strains was shown: hemolytic activity - in 64 and 36.8% of cases, DNAse activity - in 28 and 26%, respectively in clinical and wild strains. Microorganism dose, resulting in death of all the animals (LD100) did not depend on serovar of salmonella and varied from 103 to 1010 PFU/ml. Conclusion. Clinical strains were established to possess higher virulence and resistance to antibiotics compared with strains isolated from the aquatic environment.

In the review the materials on the formation ofintestinal immune homeostasis through involvement of bifidobacteria which are the key species of microbiota of human colon biotype are presented. Key function of dominant microorganisms, bifidoflora in particular, in intestinal biotype of a host is carried out by means of maintenance of self microorganisms and pronounced antagonism concerning non-self. Realization of this principle in intermicrobial relations allowed to develop algorithm of microbial self-non-self discrimination in microsymbiocenosis on the basis of detected opposite phenomenon (en-hancement/suppression) of the main physiological functions of microsymbionts survival (reproduction and adaptation) in dominant-associant pair. Primary discrimination of foreign material by bifidobacteria is the initial stage of the following «signaling» in the regulation of host immune homeostasis. Further stages of regulation occur by activation of dendritic cells by bifidobacteria with the sequential influence on differentiation of Th0 towards regulatory lymphocytes. The formation of Treg and regulation of immune homeostasis are carried out by bifidobacteria: due to direct activation of dendritic cells (ligand-receptor interactions) and maintenance of optimal cytokine balance.

A growth of pertussis morbidity is observed in many countries of the world against the background of mass vaccination. Forms of the disease course have changed. Atypical forms of pertussis occur predominately in adolescents and adults. Asymptomatic carriage of the causative agent has been established. Infection of infants with Bordetella pertussis bacteria in more than 90% of cases occurs from parents and relatives. A prolonged persistence of the causative agent has been identified. Morbidity increase in developed countries is associated with the use of acellular vaccines, that do not protect from the infection, but reduce severity of the disease. A change of genotypes of the circulating bacteria strains is observed ubiquitously. Formation of a persistent form of B. pertussis is possible due to a reversible integration of IS-elements into bvgAS operon and other virulence genes. The results of studies of invasion and survival of B. pertussis bacteria in eukaryotic cells, a change in B. pertussis bacteria population after experimental infection of laboratory mice and monkeys are presented, accumulation of avirulent insertion Bvg mutants of B. pertussis was detected. The data obtained are in accordance with the results of analysis of causative agent population in patients with typical and atypical forms of pertussis in humans. More than 50% of the population of B. pertussis bacteria in practically healthy carriers was shown to be presented by avirulent insertion Bvg mutants. B. pertussis virulence reducing as a result of inactivation of single or several virulence genes probably provide long-term persistence of bacteria in host organism and formation of apparently healthy vehicles. Follow-up studies on that front would help to formulate new attitudes to preventive measures of pertussis and lead to development of fundamentally new pharmaceuticals (vaccines) preventing formation of bacterial persistence.