Vol 88, No 6 (2011)

Aim. Study of sorption properties of various spherical polysaccharide matrixes designated as Spherocell to probiotic Lactobacillus plantarum 8RA-3 bacteria. Materials and methods. Industrial strain of L.plantarum 8PA-3 was used. The process of immobilization of lactobacilli on 3 variants of spherical sorbents was studied. The first sorbent - neutral, composed of nonpolar cellulose matrix with («0») charge, the second - DEAE obtained by modification of cellulose by diethylaminoethyl groups with positive («+») charge and the third - CM (carboxymethyl) with negative («-») charge. Cellulose matrixes were designated by us by the term Spherocell. Immobilization of bacterial cells on Spherocell was performed by addition of suspension containing 1.0x109 CFU/ml. The effect of bacterial immobilization was evaluated by CFU/ ml titration and by electron microscopy. Results. The dependence on matrix charge of adsorption immobilization on sorbent granules of lactobacilli cells was shown. At certain equal parameters (granule size, surface characteristics, charge value) the positively charged matrix sorbed 3 - 10 times more cells than neutral and 20 - 25 times more than negatively charged matrix. Each 100 - 180 pm Spherocell DEAE particle could sorb more than 1000 viable bacterial cells. Conclusion. Positively charged polysaccharide matrix Spherocell DEAE obtained by modification of cellulose by diethylaminoethyl groups is promis-больше, чем отрицательно заряженная матрица. Каждая частица Сфероцелл DEAE размером 100 - 180 мкм могла сорбировать более 1000 жизнеспособных бактериальных клеток. Заключение. Положительно заряженная полисахаридная матрица Сфероцелл DEAE, полученная путём модификации целлюлозы диэтиламиноэтиловыми группами, является перспективной для создания иммобилизованных пробиотических препаратов.

Aim. Development a method of treatment of Streptococcus pyogenes bacteria that does not disrupt the integrity of surface structure of cell and provides optimal reproducibility of enzyme preparation activity test results. Materials and methods. Museum cultures of S.pyogenes M29 and S.pyogenes T1 were used, as well as standard strain S.pyogenes T5 (ATCC) and 3 phage-associated lysine PlyC preparations (enzybiotics): 2 isolated from phage C1, third - recombinant enzyme obtained by cloning phage C1 DNA. 3 methods of S.pyogenes cells treatment were used: inactivation by chloroform, antibiotics and heating. Results. Treatment of S.pyogenes cells by rifampicin and gentamicin allows simultaneous turbidimetric determination of enzyme preparations activity and streptococci lysis effectiveness with a good reproducibility of test results. Comparison of kinetic curves of streptococci lysis killed by heating with curves of live culture lysis showed that heat treatment of cells results in a decrease of lysis depth and a reduction of enzyme activity. Pattern and effectiveness of lysis of cells incubated with chloroform approached curve of live streptococci lysis, however this method did not exclude lysis of part of cells and required presence of equipment for work with chemical substances. Conclusion. S.pyogenes test culture inactivation method by 2-step treatment of culture with antibiotics that does not disrupt the integrity of surface structure of cells and provides optimal reproducibility of enzyme preparation activity test results was developed.

Aim. Characteristics ofdrug resistance (DR) and population structure of Mycobacterium tuberculosis in Pskov region. Materials and methods. In 90 strains of M.tuberculosis drug resistance was studied by culture method and by using «TB-BIOCHIP»; genotyping was determined by spoligotyping method. Results. 55 (61.1%) of 90 M.tuberculosis strains had drug resistance, with 40 (44.4%) being multi-resistant. M.tuberculosis population was presented by SIT1 spoligotype strains of genetic families Beijing - 44.4%, LAM - 21.1%, T - 14.4%, Haarlem - 11.1% and Ural - 5.6%, according to SpolDB4. Among M.tuberculosis strains circulating in Pskov region the most widespread (44.4%) was SIT1 spoligotype (p<0.0001). DR and multi-resistant DR (MDR) in Beijing strains occurred more frequently than in «non-Beijing» strains (p<0.001 and p=0.03 respectively) and were determined by rpoB mutations Ser531^Ley and katG Ser315^Thr. All the SIT252 spoli-gotype strains were multi-resistant, and their resistance to rifampicin was determined by rpoB Asp516^Ser substitution, to isoniazid - katG Ser315^Thr and inhA_T15 substitutions. Conclusion. The data obtained gives evidence on tuberculosis epidemiological unfavorability and wide circulation of MDR M.tuberculosis strains in Pskov region.

Structure and modules of computer informational-analytical system «Electronic atlas ofRussia» is presented, the object ofmapping in this system is epidemiology of socially significant infectious diseases. Systemic information on processes of emergence and spread of socially significant infectious diseases (anthroponoses, zoonoses and sapronoses) in the population ofRussian Federation is presented in the atlas. Detailed electronic maps of country territory filled with prognosis-analytical information created by using technological achievements of mathematic and computer modeling of epidemics and outbreaks of viral and bacterial infections are of particular interest. Atlas allows to objectively evaluate the pattern of infection spread, prepare prognoses ofepidemic and outbreak developments taking into account the implementation of control measures (vaccination, prophylaxis, diagnostics and therapy) and evaluate their economic effectiveness.

Aim. Development ofmethodical approach of evaluation of microbial «friend-foe» identification in human intestine microsymbiocenosis. Materials and methods. 9 bifidobacteria cultures (dominants) and 18 opportunistic microorganism strains (associants) isolated from patients during examination for intestine dysbiosis and identified by conventional methods were used. Evaluation of microbial «friend-foe» identification in microsymbiocenosis was performed by author developed technique that is based on determination of growth factors (GF), antilysozyme activity (ALA) and formation of biofilms (BFF) of associants co-incubated with exometabolites of dominants. GF, ALA, BFF were studied photometrically (Bukharin O.V, 1999, 2009; O'Toole G.A., 2000). The data were statistically analyzed by Fisher-Student criteria. Results. The detected opposite (increase/reduction) phenomenon of the «dominant-associant» pair allowed realization of the «friend-foe» identification in microsymbiocenosis. Associants (E.coli and Enterococcus faecium) were «friend» species, in which bifidobacteria exometabolites did not change growth properties and stimulated ALA (by 17,5 - 32%) and BFF (by 25 - 39%). Associants (Escherichia coli, Klebsiella pneumoniaе, Staphylococcus aureus, Candida albicans) were «foe» microsymbiont species, in which bifidoflora exometabolites decreased GF (by 20,7 - 68%), ALA (by 22,7 - 54%) and BFF (by 22,5 - 39%). Conclusion. Indigenous microflora during microsymbiocenosis formation can participate in «friend-foe» identification, the basis of which is determined by microsymbiont exometabolites. The data obtained open a perspective of understanding mechanisms of intramicrobial interactions and can be used for both diagnostics and optimal selection of «candidates» during creation of new probiotics and synbiotics.

Aim. Study of anti-diphtheria and antitetanus immunity in women in early postpartum period depending on age. Materials and methods. Women in early postpartum period (n=139) with unknown vaccine anamnesis aged 17 to 44 years and under the supervision of Rostov-on-Don maternity hospital No. 2 were examined for the evaluation of the anti-diphtheria and anti-tetanus immunity state. Results. All the women had high level of protection form these infections. The level of anti-tetanus immunity intensity in the examined was higher than anti-diphtheria. Conclusion. Monitoring of anti-diphtheria and anti-tetanus immunity in women of childbearing age is necessary to resolve the issue of vaccine administration in this group. High level of maternal immunity intensity will allow to form a sufficient protection from infectious agents in neonates.

Aim. Evaluate the influence of electromagnetic emission (EME) at the frequencies of molecular absorption and emission spectra of atmospheric oxygen and nitrogen oxide (MAES O2 and MAES NO respectively) on the adhesion, population progress and biofilm formation of Pseudomonas aeruginosa. Materials and methods. Adhesive activity was evaluated by mean adhesion index (MAI) of bacteria on human erythrocytes. Population growth dynamic was assessed by optical density index of broth cultures; biofilm formation - by values of optical density of the cells attached to the surface ofpolystyrol wells. Results. P.aemginosa bacteria had high adhesive properties that have increased under the influence of MAES O2 frequency emission and have not changed under the influence ofMAES NO frequency. Exposure of bacteria to MAES NO frequency did not influence the population progress; exposure to MAES O2 frequency stimulated the biofilm formation ability of the bacteria, and MAES NO - decreased this ability. Conclusion. EME at MAES NO frequency can be used to suppress bacterial biofilm formation by pseudomonas.

Aim. Search of new targets and improvements of molecular-genetic detection methods of species specific DNA fragments of out-of-hospital pneumonia (OP) infection agents in sputum. Materials and methods. Species specific oligonucleotide primers to Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Branhamella catarrhalis were selected. 103 sputum samples from OP patients and 15 from practically healthy humans were test by using PCR and bacteriological method. Results. Mixed bacterial flora was detected by bacteriologic method in OP in 26 cases (25.2%). 14 patients (13.6%) had B.catarrhalis DNA in sputum, 12 patients (11.6%) - S.pneumoniaе, H.influenzae was detected in 11 examined individuals (10.7%), K.pneumoniae (7.9%) - in 8. In 32 cases (30%) etiology of OP could not be deciphered bacteriologically. B.catarrhalis was detected in 19 (18.5%) of OP patients, S.pneumoniaе - in 17 (16.5%), H.influenzaе and K.pneumoniae - in 14.5% and 12.6% (15 and 13 patients) respectively by using the developed PCR primers. In 16.5% cases (17 patients) positive results against several bacteria species simultaneously were obtained by PCR method, in 3.9% (4 patients) P.aeruginosawas detected. Conclusion. Use of PCR for detection of out-of-hospital pneumonia agents DNA significantly increases informativity of the study when compared with bacteriological method.

Aim. Study of extracellular membrane nanovesicles production by Aeromonas hydrophila and Aeromonas salmonicida bacteria on a subcellular level. Materials and methods. 4 strains ofA.hydrophila: 342-1, Е 8-8, Н 336 and Н 1-6-05 and 1 strain of A.salmonicida А-450 as well as intact Wistar line rats were used. Methods of transmission electron microscopy: ultrathin sectioning and negative contrasting were used. Results. A.hydrophila and A.salmonicida bacteria produced in pure cultures excrete into the environment extracellular membrane nanovesicles. The size of these vesicles varies from 20 to 200 nm in diameter. The process of gemmation from bacterial cell and possibility ofobtaining isolated membrane nanovesicles preparations is shown. These vesicles are detected in ultrathin sections of apical surface of intact rat intestine among accumulations ofparietal microorganisms that colonize mucous membrane. Extracellular membrane nanovesicles excreted by aeromonas are analogous by size and ultrastructure to vesicles of other species of gram-negative bacteria described in the literature. Conclusion. During production of A.hydrophila and A.salmonicida bacteria in vitro nanovesicles are formed from the outer membranes of the cells and excreted into the environment, the nanovesicles are similar to those detected in ultrathin sections of the surface of intestine of rats among accumulations of parietal microorganisms and in glycocalix between epitheliocyte microvilli.

Aim. Study of specter of low-manifest infections (LMI) with central nervous system (CNS) damage and their role in patients in prolonged unconscious state (PUS) of noninflammatory etiology. Materials and methods. 32 patients (23 male, 9 female; age 14 - 58) in PUS of various etiology were examined. The main group (18 patients) received therapy against all infectious diseases including LMI; control group (14 patients) - only against common and nosocomial microflora. Patients were immunologically, infectologically and neurologically examined in dynamic. The data obtained were treated by using STATISTICA for Windows (version 5.5). Results. Significant differences in immune and infectologic status depending on the nature ofprimary CNS damage were not detected. Immunodeficiency was detected in all patients; 94% of patients had increased non-specific IgM and IgE. Among LMI agents Chlamydia spp. were predominant. Cultural and/or PCR methods detected this microorganism during the primary examination in cerebrospinal fluid samples in 56% patients and in blood samples in 56%; during the second diagnostics or autopsy-only in 13 and 25%, respectively. Detection of Bacteroides fragilis, Human Herpes Virus (HHV-6), Virus Epstein Barr (VEB), Cytomegalovirus (CMV) in cerebrospinal fluid, blood and on mucous membranes of nasopharynx and conjunctiva was grouped more frequently with the presence of Chlamydia spp. in the CNS (p<0.05) than with other LMI agents. Sanation of CNS from LMI was significantly accompanied by regeneration ofcommunicative activity in comparison with the control group. Conclusion. In patients with PUS high frequency of CNS infection by various LMI agents and primarily Chlamydia spp. should be considered. Sanation from LMI can become a «window» for effective neuro-regenerative treatment.

Aim. Detection of features of qualitative and quantitative composition of large intestine microbiocenosis of humans with various degree of local metabolic disorders during dysbacterioses due to various causes. Materials and methods. Microflora oflarge intestine and content of malonic dialdehyde (MDA) in coprofiltrates of 330 adult humans with large intestine dysbacterioses due to various causes were studied. Results. It was established, that high MDA content in coprofiltrates matches higher quantities of opportunistic microorganisms and atypical escherichia in microflora composition of large intestine. Relation of MDA composition in coprofiltrates and factors that cause dysbacteriosis were not detected. Conclusion. The studies performed give evidence that changes in local metabolic activity may be a single mechanism of development of large intestine dysbacterioses that are caused by various factors.

The review is dedicated to the role of sensory nerve endings of the gut, vegetal and central nervous system (CNS) in the diseases of gastrointestinal tract. Molecular-cellular inter-relations of nerve endings of the gut and neurons of the CNS are a key axis that among with neuroendocrine and immune responses, define the clinical manifestation and rehabilitation potential of the patient in the development of infectious process in the gut. Infectious-inflammation processes in the gut of various etiologies promote the increase of permeability of the intestine barrier with consequent trans-intestinal translocation of toxins and molecular mediators of inflammation to the system bloodstream. Bacterial toxins including LPS and cytokine imbalance induce microglia damage that defines destabilization ofthe barrier and vulnerability of neurons. The consequence is the inadequate reaction from autonomous nervous system with the development of uncontrolled abdominal spasms and increasing muscular atrophy. Toxemia at the same time promotes the increase of hematoencephalic barrier permeability, intake of inflammatory cytokines into the brain that induce inflammation in the brain periventricular areas with the development of intestinal encephalopathy. The assumed pathogenetic mechanism dictates a new therapy strategy that is mainly directed at brain protection: administration of etiotropic and anti-inflammatory drags, myotropic spasmolytics and various neuroprotectors.

Chlamydia trachomatis is one of the most widespread bacterial pathogens in the world that is transmitted sexually. Thereby a development of vaccine preparation for therapy and prophylaxis of infections caused by C.trachomatis is an actual topic of scientific research across the entire world. These vaccines could be used for both prophylaxis and therapy of already established chlamydia infection and, respectively, reduce the risk of chronic condition and prevent the spread of pathogen in the population. Features of chlamydia biology that are associated with obligate intracellular parasitism and im-munopathologic state induced by this agent significantly complicate developments in this field. Currently a vaccine preparation that has reached clinical trials does not exist.