Vol 86, No 4 (2009)

Features of meningococcal carriage as a form of microorganism's persistence necessary for survival and species preservation are discussed in this review. New data on genetic heterogeneity of meningococcal population, which is major determinant of occurrence of asymptomatic forms, are presented. Process of formation of meningococcal biofilms is described. Materials about systemic and local immunity developed during meningococcal carriage and possible influence of vaccination on carriage rate are reviewed. Role of persistence of meningococci in stimulation of immune protection of population from severe forms is discussed.

Aim. To study the influence of associative microflora exometabolites on enzymatic activity and persistence potential of Staphylococcus aureus during a day. Materials and methods. Collection strains S.aureus ATCC 25923, Escherichia coli 35218, and Pseudomonas aeruginosa ATCC 27853 were used. Exometabolites of E.coli and P.aeruginosa were obtained from broth cultures, which were centrifugated and supernatant was separated and sterilized by addition of chloroform. Exometabolites of microorganisms-associates were mixed with nutrient broth in 1:10 ratio with subsequent addition of pure culture of S.aureus . Broth cultures of staphylococci which do not contain exometabolites were used as controls. Biorhythms of proliferative activity of microorganisms were determined using original method developed by the authors. Catalase, hemolytic, and antilysozyme activities of bacteria were determined by photometric method, whereas activity of protease — by biuretic method. Results were statistically processed by Student's test. Results. Analysis of experimental data on the influence of exametabolites of E.coli and P.aeruginosa collection strains on daily dynamics of activity of aggression and persistence enzymes of S.aureus revealed daily cycles of studied parameters and desynchronization of biorhythms. Conclusion. Use of the developed model system allows to reveal regulatory effects of associated microorganisms on biological (pathogenic) characteristics of causative agent. Bacterial interrelations are significant regulatory action on pathogenic/persistence characteristics of a pathogen which should be taken in account for pathogenic assessment of an agent during infection it caused.

Aim. To assess antipersistence characteristics of synthetic antioxidants belonging to class of triterpenoids. Materials and methods. Synthetic antioxidants synthesized in the Institute of Organic Chemistry of Russian Academy of Science and belonging to class of triterpenoids were used in the study. Antioxidant activity of compounds was assessed by amperometric method using analyzer «Tsvet Yauza-AAA-01» («Chimav tomatika Ltd.», Moscow) and expressed in quercetin units (QU). Influence of synthetic antioxidants on persistence characteristics (antilysozyme, anticomplement, and anticarnosine activities) of Klebsiella pneumoniae 278 (Tarasevitch State Institute of Standardization and Control for Immunobiologic Preparations) and Staphylococcus aureus was determined by conventional methods. Results. Antioxidant activity of studied compounds varied from 1.53 QU to 11.82 QU. Compound No. 5, which had maximal antioxidant activity and was characterized by presence of 2 atoms of oxygen and hydroxyl radical in its structure, influenced on expression of persistence characteristics of studied microorganisms most efficiently. Conclusion. Obtained results could be used for the development of new drugs aimed against persisting pathogens.

Aim. To select the most susceptible line of mice which allows to conduct comparative studies of infectious process caused by different strains of B.cepacia in order to explore correlation between ability to form biofilms and persistence of bacteria in organs of infected animals. Materials and methods. Strain B.cenocepacia 370, which is a clinical isolate, and its mutants with modified ability to form biofilms were used. Conditional microbiologic methods and biological models of intraperitoneal and intranasal inoculation of mice belonging to 4 lines: BALB/c, BLACK, I/St, and A/Sn derived in Central Institute of Tuberculosis were employed. Criteria of persistence was duration of isolation of different strains of bacteria from lungs and spleen of inoculated animals as well as number of CFU. Results. The most susceptible line of mice which enables to conduct comparative studies of infectious process caused by Burkholderia species was determined. It was shown that even after intraperitoneal inoculation the agent was better preserved in lungs than in spleen that corresponds to natural localization of this infection. At any time of observation the number of cells of mutant strain, which is a superproducer of biofilms, isolated from organs of inoculated mice was 2 — 10 times higher than number of isolated cells of mutant, which do not produce biofilms. Conclusion. Correlation of more prolonged persistence of B.cenocepacia in organs of inoculated animals in vivo with ability of the agent to form biofilms determined in vitro is experimentally established. The susceptible line of mice which allows to conduct comparative studies of dynamics of infectious process caused by various strains of Burkholderia species was revealed. It was shown that irrespective from method of inoculation B.cepacia are able to continuously persist in organism of susceptible animals with lungs as a predominant localization.

Aim. To study influence of chemically synthesized lactones with different length of carbonic chain on formation of biofilms by Burkholderia cepacia and Pseudomonas aeruginosa . Materials and methods. Fifty-four strains of B.cepacia including reference strains and clinical isolates as well as etalon strain PA103 of P.aeruginosa were used. Lactones C4 [N-(3-hydroxybutanoyl-L-gomoserine lactone], C6 [N-(3-oxoohexanoyl)-L-gomoserine lactone], C8 [N-(3-oxooctanoyl)-L-gomoserine lactone], C0 [L-gomoserine lactone-HBr] were synthesized and purified by column chromatography. Formation of biofilms was studied by determination the ability of B.capacia strains to adhesion on the surface of 96-well polystyrene plate. Results. Ability to biofilms formation was identified in 83.3% studied strains. It was shown that lactones C4+C8 and C6+C8 when added to cultivation medium improve growth of B.cepacia biofilm. Analysis of optical density (OD) values for P.aeruginosa biofilm revealed that lactones C4, C8, C4+C6, C4+C8, C6+C8 inhibit the formation of biofilm. The most prominent decrease of P.aeruginosa biofilm OD was observed during growth of culture in presence of C0. Conclusion. Obtained data point to different effects of lactones and their combinations on formation of biofilms by B.cepacia and P.aeruginosa . Suppression of biofilm formation by P.aeruginosa induced by lactone C0 confirms the need for development of new bacteriostatic drugs, which will be able to inhibit function of the «quorum sensing» regulatory system.

Aim. To assess influence of bifidobacteria's exometabolites on expression of antilysozyme activity of microflora and its ability to biofilm formation. Materials and methods. Six strains of bifidobacteria belonging to B.longum , B.bifidum , and B.adolescentis as well as 42 strains of opportunistic microorganisms belonging to Escherichia coli , Klebsiella pneumoniae , Enterobacter cloacae , Staphylococcus aureus , Candida albicans , and Rhodotorula rubra isolated by conventional methods during assessment of intestinal microbiocenosis in humans. In order to study the influence of bifidoflora's exometabolites on associative flora, obtained filtrates were mixed with nutrient broth in 1:10 ratio with subsequent introduction of pure culture of opportunistic microorganism. Nutritional broth was used instead of filtrates of bifidobacteria for obtaining the control samples. Formation of biofilms was determined on the ability of strains of microorganisms to adhere on the surface of 96-well polystyrene plate, whereas the antilysozyme activity was studied by photometric method. Results. Performed studies revealed multidirectional but mostly inhibiting effect of bifidobacteria's metabolites on antilysozyme activity of opportunistic microorganisms and their ability to form biofilms. Changes of these characteristics was observed both in Gram-positive and Gram-negative bacteria as well as in yeast fungi and depended from species of bifidobacteria and associative opportunistic microflora. Conclusion. Obtained data open perspective for search of control on infectious process on the basis of modification of persistence characteristics of associative pathogens and their ability to form biofilms in host's biotope.

Aim. To study the influence of Solcotichovac vaccine on formation of neutrophil extracellular traps. Materials and methods Solcotrichovac vaccine administered in various doses was used in order to assess the formation of neutrophil extracellular traps in peripheral blood of women of childbearing age. Results. Therapeutic dose of Solcotrichovac stimulated the most prominent formation of neutrophil extracellular traps. Conclusion. Solcotrichovac vaccine stimulated non-specific arm of mucosal immunity due to formation of neutrophil extracellular traps and it is probable that this mechanism explains the effect of this vaccine.

Aim. To study the effect of active forms of oxygen on bacterial population structure on antilysozyme characteristic. Materials and methods. Antilysozyme activity of bacterial clones obtained after their treatment by hydrogen peroxide and hydroxyl radical in sublethal (LD 10) and non-lethal concentrations. Results. Shift of bacterial population structure in direction to increased proportion of clones with high level of antilysozyme activity with elevating average population level of this characteristic under the influence of sublethal (LD 10) concentrations of hydrogen peroxide and hydroxyl radical was established. Using of non-lethal concentrations of active forms of oxygen resulted in decrease of average population level of antilysozyme activity and emergence of clones with lower level of expression of this characteristic than in control population. Conclusion. It was assumed that active forms of oxygen provide variety of types of interaction of microorganisms with each other and with macroorganism due to change of intrapopulation heterogeneity of biological characteristics of bacteria, which enables them to realize their persistence potential.

Definition of endogenous bacterial infections (EBI) is formulated as well as their nosological variability is demonstrated and clinical and microbiological characteristics are characterized. Universal scheme of pathogenesis of these infections with separation of key phases of their course: premorbid phase, colonization, alteration, sanation or persistence. Important role of persistence characteristics of microorganisms for the development of EBI, which provide survival of agents during contacts with humoral and cellular effectors of host's immunity, is substantiated.

Aim. To determine overall number as well as number of viable cells in continuously incubated cultures of E.carotovora by methods of confocal microscopy and quantitative PCR-analysis. Materials and methods. Strain E.carotov ora atroseptica SCRI1043 was grown on LB medium to density 2x10 9 CFU/ml. Cells were aggregated by centrifugation and transferred on fresh LB medium, containing alkyloxybenzol, or on the AB medium, which was deficient on phosphorus and carbon. BacLight LIVE/ DEAD kit in combination with confocal laser microscopy as well as quantitative PCR were used for the determination of the number of viable cells. Results. Total number and number of viable cells in cultures on AB medium was high (10 8 — 10 9 and 10 7 — 10 8 cells/ml respectively) up to 3—5 months of cultivation. Though, number of cultivated cells significantly decreased in all variants of the experiment. Number of viable cells in such cultures was several orders greater than genomic copies detected by PCR. Efficacy of DNA amplification increased after dialysis and deproteinization of samples. Conclusion. Loss of cultivation ability when number of viable bacteria is high points to possible switch of E.carotovora cells in non-cultivated state under unfavourable conditions. We assume that it is accompanied by formation of low-molecular components and DNAbound proteins in cells, which inhibit PCR.

Aim. To study the time of preservation of antigens and DNA of urogenital mycoplasmas in circulating immune complexes (CIC) in blood of rabbits after single inoculation. Materials and methods. Rabbits were inoculated with Mycoplasma hominis and Ureaplasma urealiticum cell cultures washed in fetal calf serum. Reaction of aggregate-hemagglutination, immunofluorescence assay and PCR were used for detection of mycoplasmas' antigens and DNA. Results. It was shown that DNA and antigens of M.hominis persist in free state and in structure of CIC during 1 month and 3 months respectively. In immunized rabbits antigens and DNA of mycoplasmas were detected in CIC structure even 6 months after the last immunization. Pattern of detection of DNA and antigens of U.urealyticum in blood of inoculated rabbits consists in that both DNA and antigens of the microorganism were detected in structure of CIC in blood samples during 70 days, whereas in free state they were detected only during 35 days. Incomplete elimination of CIC is possibly related to their small size (11S and lower) that allows them to circulate for a long time. Conclusion. Prolonged persistence of antigens and DNA of mycoplasmas in CIC structure is a fact that requires refinement of diagnostic criteria used for control of effectiveness of etiotropic therapy.

Aim. To study the state of vaginal microbiocenosis in women with endometritis and to assess association between microecological disturbances and characteristic of clinical course of the disease. Materials and methods. Two hundred women of childbearing age with endometritis were studied, of which 83 had acute form of the disease, and 117 — chronic form. Control group was comprised by 30 clinically healthy women. Antilysozyme, anticomplement, and antilactoferrin activities were determined in isolated and identified microorganisms. Microecological disturbances in vaginal biotope were determined applying criteria proposed by Sokolova K.Ya. et al. and Amsel R. et al. Results. Microecological disturbances in vaginal biotope were revealed on women with endometritis — in particular, bacterial vaginosis, deficiency of dominant microflora (lactobacilli), and the presence of associate microorganisms with clearly expressed persistence characteristics. On the background

Aim. To assess prevalence and intensity of antilactoferrin and sIgA-protease activity in strains of staphylococci and to study the state of local immunity in resident and transitory bacterial carriers. Materials and methods. Strains of Staphylococcus aureus and S.epidermidis isolated from 67 healthy persons, who were tested for staphylococcal carriage with subsequent differentiation of types of carriage according to Chistovitch's method, were studied. Antilactoferrin and sIgA-protease activity as well as levels of lactoferrin and sIgA in nasal secretions were determined using fluoroimmunoassay. Results. Higher prevalence as well as intensity of antilactoferrin and sIgA-protease activity and combination of these characteristics was determined for strains of S.aureus from resident carriers compared with strains from transitory carriers. Higher levels of lactoferrin and sIgA were observed in nasal secretions of resident carriers compared with transitory carriers. Conclusion. Studied factors could be the markers of resident type of carriage that should be considered for the development of effective measures for sanation in persons with prolonged bacterial carriage.

Aim. To study virulence of Toxoplasma gondii strain isolated from pathologicoanatomic brain tissue of HIV-infected patients on in vivo model as well as immune response to the pathogen in immunocompetent animals and in animals with cyclophosphamide-induced areactivity. Materials and methods. Groups of immunocompetent and immunodeficient mice were inoculated with cysts of T.gondii obtained from brain tissues of deceased HIV-infected patients. Parasites were detected in different organs and tissues by PCR as well as by parasitological and histological methods. Antibodies to T.gondii belonging to isotypes IgG, IgG1, IgG2, IgA were detected by immunofluorescence assay. Results. Cysts of the pathogen located predominantly in animals' brain, and more rarely — in spleen and liver. Using detection of antibodies of different isotypes, which marked stage of invasion, dynamics of humoral response during persistence of toxoplasms in organism was determined. Analysis of disease pathogenesis as well as peculiarities of immune response to the pathogen in immunocompetent animals and in animals with cyclophosphamide-induced areactivity was performed. Conclusion. Characteristics of strains of toxoplasms isolated from material of brain section of patients with HIV infection were studied. Their low virulence and ability to prolonged persistence in organism of mice were demonstrated on in vivo model. Possible association of T.gondii strains' virulence and clinical symptoms with pathogen ' s genetic polymorphism and its clonal population structure was discussed.

Aim. To determine the ability of agents of surgical soft tissue infections to form biofilms. Materials and methods. Material for the study was strains of microorganisms causing surgical soft tissue infection (phlegmon): 38 strains of Staphylococcus aureus , 16 strains of coagulase-negative staphylococci, 20 strains of enterococci, and 10 strains of pseudomonades. Ability of microorganisms to form biofilms was determined by photometric method. Microscopic evaluation included study of pathologic material from wounds as well as biofilms obtained in vitro . Results. All strains of pseudomonades and 80% of enterobacteria strains had marked ability to form biofilms. This characteristic was less prevalent and expressed in staphylococci. Direct relation between ability of microorganism to form biofilm and duration of illness (correlation coefficient — 0.68) was revealed. Biofilms in wounds were detected only 3 or more days after surgery. Conclusion. Agents of surgical soft tissue infections are able to form biofilms. This ability of microorganisms realizes not only in vitro but in wound too. Direct correlation between expression of ability to form biofilms and duration of illness allows to consider this characteristic as marker of switch of infectious process to chronic state.

Role of effects of technological factors on microflora of food products during process of their manufacture is discussed. Influence of physical, chemical and biological stress actions on formation of several types of tolerability of bacteria contaminating food products is presented. Regulatory mechanisms of response reactions of microorganisms on adverse actions, patterns of behavior of pathogenic bacteria in stress circumstances, relationship between induced stress tolerability and level of expression of virulent characteristics of microorganisms are discussed. Necessity to study stress responses by discovering full genetic and proteomic profiles of pathogenic bacteria is substantiated.