Vol 89, No 4 (2012)

Primary results of study of problem of microorganism persistence over the last 2 decades on 7 All-Russian conferences in Orenburg are examined in the article. Milestones of both fundamental research and practically significant studies are designated, the role of persistent potential of microorganisms in infectious pathology is evaluated. The emerging turn of studies from persistence to symbiosis is consonant with the idea of international project Human microbiom and allows to use the persistent potential of microorganisms as one of the instruments of resolving issues of infectology.

Aim. Evaluate influence of mutation of Listeria monocytogenes genes coding murein-tetrapeptide L,D-carboxypeptidase Lmo0028 and Lmo1638 on dynamics of infectious process and interaction of purified muropeptides with NOD1 receptor. Materials and methods. Wild type EGDe strain and recombinant strains GIMins1638 и GIMins0028 obtained on its basis by site-specific mutagenesis were used. Infectious process dynamics was studied on the model of intravenous infection of BALB/c mice. Ligand-receptor interaction activity of muropeptides isolated from recombinant and parent strains were assayed on HEK293-hNOD1 cell line expressing NOD1 receptor and containing in their genome ƒ-galactosidase reporter gene under the control of NF-kB dependent promoter expression. Results. Lack of Lmo0028 decelerates reproduction of listerias in animal liver starting from 24 hours and at later terms after the infection whereas lack of Lmo1638 leads to increase of microbial load 6 and 24 hours after the infection with no influence on further infection. Differences in activation of NOD1 receptor by muropeptides isolated from recombinant and parent strains were not detected. Conclusion. Despite high homology murein-tetrapeptide L,Dcarboxypeptidase Lmo0028 and Lmo1638 make a different contribution to the development of infectious process caused by L. monocytogenes in BALB/c line mice. Lack of differences in NOD1 receptor activation may be associated with compensation of enzymatic functions in strains with mutation in each of the genes owing to the presence of homologous protein.

Aim. Develop in vitro model for studying production of cytokines by monocyte cells infected with Chlamydia trachomatis mediated by type III secretion system (TTSS). Materials and methods. Strain C. trachomatis L2/434/Bu was used in the experiments, culture of human monocytes U-937 was infected by this strain. Level of inflammatory cytokines was measured on flow analyzer Bio- Plex 200 (Bio-Rad Laboratories). Low molecular compound LHC-342 which belongs to the class of heterocyclic compounds was used as TTSS inhibitor. Results. 24 hours after the infection with C. trachomatis culture 8 analyzed cytokines are induced in U-937 cells (IL-1ƒ, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-ƒ, TNFƒ). The most pronounced increase was observed for IL-8, GM-CSF and IFN-ƒ. Introduction of TTSS inhibitor into the culture of infected cells suppressed chlamydia growth, but addition of FeSO4 restored the growth of chlamydiae. And activity associated with translocation of effector TTSS protein IncA to inclusion membrane was suppressed. Under the conditions of the obtained model of TTSS inhibition during intracellular development of C. trachomatis a significant decrease of 2 pro-inflammatory cytokines - IL-6 and IL-1ƒ - was observed. Conclusion. Cytokine response plays a key role in the protective immune response in chlamydia infection but at the same time induces immunopathologic conditions. The data obtained give reasons to assume role of C. trachomatis TTSS in the induction of this component of immune response that requires further detailed studies.

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 of dysbiotic 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.

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.

Aim. Study the influence of exometabolites of B. bifidum on biological properties of bacteria that are the members of normoflora and their ability to interact with associative microsymbionts. Materials and methods. Bacterial strains that are members of the normal microflora of human intestine: B. bifidum, Lactobacillus acidophilus, Enterococcus faecium and Escherichia coli lactose positive non-hemolytic (lac +/ hly -) were used. As opportunistic microorganisms cultures of E. coli lactose negative hemolytic (lac -/hly +), Klebsiella pneumoniaе and Staphylococcus aureus were used. Isolation and identification of microorganisms was performed by generally accepted methods according to guidances. In the first series of experiments influence of B. bifidum metabolites on biological properties of microorganisms that are members of normoflora was studied. In the second series - the influence of bifidobacteria supernatants on interrelations of B. bifidum, L. acidophilus and E. coli lac +/hly - with opportunistic associants. Growth properties (GP), biofilm formation (BFF) and anti-lysozyme activity (ALA) of microorganisms was studied photometrically. Optical density measurement were performed on ELx808 (BioTek, USA) photometer. The data obtained were treated by nonparametric method using Mann-Whitney criteria. Results. B. bifidum supernatant was established to stimulate in 33.3 - 66.7% of cases or did not alter growth/reproduction, BFF and ALA of microorganisms that are characteristic for eubiosis of intestine including bacteria of the same species that could have implications for realization by bifidobacteria of biotope colonization resistance. Features of interaction of exometabolites of bifidobacteria with microorganisms that are characteristic for eubiosis of human intestine consisting in enchantment or changes of effects of the influence of normoflora members on BFF of associants were revealed. The maximum enchantment of inhibitory effect of indigenous strains under the influence of bifidobacteria was noted in associations E. coli lac +/hly - - E. coli lac -/hly + as well as E. faecium - S. аureus. Conclusion. Thus, the data obtained may be used for detection of mechanisms of functioning of normal microsymbiocenosis in human associative symbiosis.

Aim. Study prevalence and intensity of persistent properties in bacteria inhabiting highly mineralized water bodies and determine their role in interaction with halophilous heterotrophic protozoa. Materials and methods. 300 bacteria strains and 3 cultures of heterotrophic protozoa isolated from water bodies with mineralization of 2 - 350 g/l were studied. Antilysozyme (ALA), antihistone (AHA) activity of bacteria, protozoa lysozyme were evaluated by dish and photometric methods. Protozoa histones were evaluated cytochemically. Interaction of protozoa and Escherichia coli was evaluated by experimental co-cultivation. Results. Presence of lysozyme and histones was shown in halophilous heterotrophic protozoa. Prevalence of ALA and AHA in bacteria was shown to increase as water body mineralization decreases. Intensity of E. coli elimination from brine was determined to depend on the bacteria ALA level and phagocytic activity of protozoa. Participation of halotolerant protozoa in formation of heterogeneity of bacterial population by ALA was shown. Conclusion. In biocenoses of highly mineralized water bodies functioning of lysozyme-antilysozyme, histone-antihistone systems was shown. Bacteria with high persistent potential may impair sanitary parameters of highly mineralized water bodies, process of self-purification of which depends directly on phagocytic activity of protozoa.

Significance of symbiotic relations formed by associative symbiosis type for autochthonous and allochthonous microflora of natural water bodies is shown. Generality of symbiotic interaction mechanisms of symbionts in limnetic and halophilous communities provided by secreted factors of natural resistance from the side of the host, and by factors of persistence from the side of symbionts is proven based on a set of examples. Features of operation of lysozyme-antilysozyme, histon-antihiston, hydrogen peroxide-catalase functional systems in symbiotic interactions of autotrophic and heterotrophic components of hydrobiocenosis with dominant and associative microflora are presented. Associative microflora of allochthonous origin was shown to actively use the ecologically formed system of interaction between hydrobionts that facilitates survival of these microorganisms and preservation of their persistent potential, and as a result leads to biocenosis disorders. The knowledge obtained open new possibilities and perspectives of research of sanitary and ecological aspects of vital activity of aquatic biocenoses.

The article is dedicated to examination and analysis of materials on translocation of microflora and its products from intestine to the internal environment of the macroorganism and persistence of biologically active substances of microflora in the bloodstream. High frequency of translocation and persistence of intestine microflora and its components in system bloodstream is shown. Persistent biologically active substances of endogenous microorganisms take part in human physiology and pathology.

Aim. Study features of persistence of Burkholderia cepacia in mucoviscidosis patients. Materials and methods. In the period from 2008 to 2009, 56 B. cepacia strains isolated from children with mucoviscidosis were obtained. 114 medical histories of children with mucoviscidosis from various age groups were analyzed. The developed algorithm for identification and typing including phenotype and molecular biology methods was used to identify B. cepacia bacteria. Strain genotyping was carried out by RAPDPCR with random oligonucleotide primer as well as pulse-electrophoresis. Results. Persistence of associations of microogranisms in 59.4% of cases was established to be the feature of persistent infection in mucoviscidosis. The feature of persistence of B. cepacia strains in patients with diagnosis of mucoviscidosis mixed form, severe course is persistence in association with Pseudomonas aeruginоsa. B. cepacia bacteria that can persist in mucoviscidosis patients are characterized by resistance to many antibiotics. A prolonged (up to 1 year and 5 months) persistence of B. cepacia strains isolated from 1 patient was proven by using microflora monitoring of lower respiratory tract. Conclusion. B. cepacia bacteria may colonize lower respiratory tract of mucoviscidosis patients, persist for a long time and be transmitted between patients.

Aim. Study previously unknown forms of persistence of Mycoplasma hominis in host organism. Materials and methods. Culture method was used for detection of mycoplasmas. Identification was carried out by serological, electron microscopy methods, classic PCR and real time PCR; circulating immune complexes (CIC) were isolated by PEG precipitation. Results. Classic micoplasma cultures could not be isolated from blood even once. At the same time mini-colony cultures composed of mini-cells that were hardly passaged but sometimes formed continuous layer of the same colonies were isolated from blood serum samples with high frequency. During reseeding for more than 1 year they never acquired classic form. Not only antigens of M. hominis but its DNA were shown to be present in CIC. Viable cells forming mini-colonies identical to those isolated from blood sera were isolated from circulating immune complexes. A system of evidence on identity of isolated M. hominis cultures is presented. Cultures had infectivity and an ability to persist in organs of experimentally infected mice. Conclusion. The isolated forms are apparently the result of adaptation of mycoplasmas to humoral immunity factors.

Aim. Study the expression of cytokines by vaginal epitheliocytes in the process of interaction with dominant and associative microsymbionts. Materials and methods. IL-8, IL-6, IL-1ƒ and TNFƒ expression in response to interaction with heat inactivated Lactobacillus spp., Staphylococcus aureus, Escherichia coli, Corynebacterium spp. or their secretory products in comparison with basal expression of cytokines by vaginal epitheliocytes was studied. Results. Lactobacilli secretory products were shown not to influence the expression of IL-8 and IL-1ƒ and moderately stimulated IL-6 and TNFƒ expression. Contact of epitheliocytes with heat inactivated lactobacilli increased secretion of IL-8, IL-6 and IL-1ƒ and reduced TNFƒ production. Secretory products of S. aureus and E. coli caused stimulation of IL-6, IL-1ƒ production and practically did not change the expression of IL-8 and TNFƒ. Contact of epitheliocytes with heat inactivated S. aureus suppressed TNFƒ production and had no influence on IL-8, IL-6 and IL-1ƒ expression, contact with E. coli stimulated TNFƒ and IL-1ƒ expression and suppressed IL-6 expression. Changes in cytokine expression during interaction of epitheliocytes with corynebacteria were largely similar to the results of interaction with lactobacilli except IL-6 production that was markedly stimulated by corynebacteria secretory products. Conclusion. In epithelial-bacterial interactions dominant and associative microorganisms have a differential effect on functional status of mucosal epitheliocytes manifesting in production of cytokines that could be the basis of mucosal immunity regulation.

Aim. Determine the role of opportunistic infections causative agents in ethology of obstructive bronchitises and prolonged subfebrilities in children. Materials and methods. 56 children with the diagnosis of obstructive bronchitis and 46 children with the diagnosis of prolonged subfebrility were examined for the presence of herpes, mycoplasma and pneumocystic infections. EIA, IIF, rapid culture method, PCR were used. Results. The highest number of cases of mixed infection was detected in children with HHV-6 infection. Mixed infection was diagnosed 6 times more frequently in children with obstructive bronchitis and 9 times in children with prolonged subfebrility. The number of children with pneumocystosis in combination with other infections was 2.4 and 2 times higher than with monoinfection; with CMV infection - 4 and 2 times; with HSV infection - 5 and 4 times; EBV infection - 6 and 3.7 times. The only exception was mycoplasmosis detected in children with obstructive bronchitis where the difference between the number of mono and mixed infection cases was insignificant. Conclusion. The data obtained give evidence of wide spread of opportunistic infections.