[Infectious genesis of nephroliths (electron-microscopic study)].

Scanning electron microscopy and x-ray microstructural analysis were employed in the study of nephroliths from patients suffering from nephrolithiasis. Bacterial biofilms, urease producing microorganisms, alkaline reaction of the urine are basic factors for local urine crystallization, formation of the base of the nephroliths and its rigid fixation to the pelvic mucosa. Mechanic trauma of the pelvic tissues by the concrement results in destruction of the pelvic mucosa epithelium at the site of the nephrolith. Subsequent inflammation in the underlying connective tissue contributes to formation of connective tissue commissures fixing the conrement in the kidney. It is shown that bacteria as a part of a biofilm are capable to persist in nephroliths for a long time. Destruction of the stones during operation or lithotripsy can trigger activation of growth of bacteria integrated in the biofilm and cause septic complications. Preservation of commissures with elements of the destroyed stone after lithotripsy or surgical removal is one of the leading causes of recurrent nephrolithiasis.

[Renal calculus microflora in urolithiasis and search for agents of control of biofilms formed by uropathogenic bacteria].

AIM: Study bacterial biofilms in native material (renal calculus) by electron microscopy method and developmeit of biofilm model by isolates in vitro on sterile calculi of various chemical composition. MATERIALS AND METHODS: Bacterial spectra of microflora of renal calculus lavages were studied, isolated pure cultures were identified up to species. Comparisons of urine microflora obtained before operation in patients with urolithiasis with microflora of removed renal calculi were carried out. RESULTS: Urease activity and genes coding pathogenicity factors were detected, and the ability to form biofilms by isolates was studied. Model of formation of biofilms in vitro on sterile renal calculi was developed and candidate agents reducing the biofilm forming ability were tested. CONCLUSION: Uropathogenic microorganisms infecting renal calculi and forming biofilms on them not only support chronic infection by increased resistance to therapy but also facilitate novel lithogenesis.

[Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones--signal molecules of Quorum Sensing regulation].

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

[Biofilms of pathogenic bacteria and their role in chronization of infectious process. The search for the means to control biofilms].

The role of biofilms in the environment and in the hosts of pathogenic bacteria has recently attracted much attention of researchers. Microorganisms form biofilms at any biotic and abiotic surfaces and thereby cause serious problems in medical practice and other areas of human activity. Biofilms have been shown to be pathogenetic factors responsible for chronization of infectious process. The data are presented illustrating ubiquitous nature of biofilms, their structural and functional characteristics, and modern methods for the study of microbial communities. The discussion is focused on the role of biofilms in chronization of infectious process, enhanced resistance of biofilm organisms to antibiotics and its underlying mechanisms. Approaches to the search for new means for biofilm control during chronic infections are considered.

[Bacterial biofilms as a natural form of existence of bacteria in the environment and host organism].

Advances in microscopic analysis and molecular genetics research methods promoted the acquisition of evidence that natural bacteria populations exist predominately as substrate attached biofilms. Bacteria in biofilms are able to exchange signals and display coordinated activity that is inherent to multicellular organisms. Formation of biofilm communities turned out to be one of the main survival strategies of bacteria in their ecological niche. Bacteria in attached condition in biofilm are protected from the environmental damaging factors and effects of antibacterial substances in the environment and host organism during infection. According to contemporary conception, biofilm is a continuous layer of bacterial cells that are attached to a surface and each other, and contained in a biopolymer matrix. Such bacterial communities may be composed of bacteria of one or several species, and composed of actively functioning cells as well as latent and uncultured forms. Particular attention has recently been paid to the role of biofilms in the environment and host organism. Microorganisms form biofilm on any biotic and abiotic surfaces which creates serious problems in medicine and various areas of economic activity. Currently, it is established that biofilms are one of the pathogenetic factors of chronic inflection process formation. The review presents data on ubiquity of bacteria existence as biofilms, contemporary methods of microbial community analysis, structural-functional features of bacterial biofilms. Particular attention is paid to the role of biofilm in chronic infection process formation, heightened resistance to antibiotics of bacteria in biofilms and possible mechanisms of resistance. Screening approaches for agents against biofilms in chronic infections are discussed.

[Development of pulmonary chlamydia infection in inbred mice lines differentiated by genetically determinated sensitivity to tuberculosis infection].

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p < 0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

[Role of salmonella in biliary lithogenesis].

By the methods of light microscopy and immunocytochemistry studies of interaction between S. typhimurium and corpuscular biliary components was investigated in experimental model "bile-bacteria" It was shown that the results of this interaction was bacterial-biliary sludge formation. Bacterial extracellular mucopolysaccharides matrix and flagella's play crucial role in mechanism of sludge formation.

[Persistence of Burkholderia cenocepacia bacteria in vivo in dependence of their ability to form biofilms].

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.

[Using real-time polymerase chain reaction for detection of Legionella in objects of the environment].

AIM: To assess efficacy of using the method of quantitative detection of Legionella in objects of the environment by real-time polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: For the development of the assay, genus-specific primers from gene coding 16S rRNAas well as species-specific primers for detection of Legionella pneumophila on the basis of mip gene sequence. For quantitative detection of L. pneumophila calibration samples of pGEM plasmid containing fragment of the mip gene in known concentration were used. Samples of water and biofilms obtained from cooling stacks of production plants, systems of autonomic water supply, humidification blocks of centralized systems of air conditioning were studied. RESULTS: Correlation of results obtained with RT-PCR and bacteriologic methods was shown during monitoring of potentially dangerous water objects as well as during epidemic outbreak of Legionella infection. Importance of samples preparation stage, during which considerable losses of DNA and inhibition of reaction could occur, is underlined. Disinfection measures on the studied objects significantly influenced on the results of the RT-PCR and can lead to false positive results. CONCLUSION: Obtained results confirm usefulness of testing of potentially dangerous water objects on the presence of Legionella based on the preliminary screening with RT-PCR for the 24 hours followed by bacteriologic testing of samples for 8 - 12 days.

[Legionella natural biofilms and their role in epidemiology of the infection: methods of study and modelling].

Ability of Legionella species to form biofilms in association with other microorganisms is the key factor of their spreading in potentially dangerous water systems. Ability of different strains of Legionella to form monospecies biofilms as well as biofilms in association with Pseudomonas aeruginosa in constant conditions was analyzed. It was shown that ability of Legionella strains to form monospecies biofilms correlates with their ability to persist in biofilms formed by P. aeruginosa.

[Assessment of bactericidal activity of disinfectants against Legionella on the biofilm model].

Comparative assessment of bactericidal activity of different disinfectants against Legionella biofilms was conducted. Monospecies biofilms of 3 strains of Legionella pneumophila obtained on plastic plates in stable conditions were used as models. It has been shown that for degradation of biofilms as well as for prophylactic action of disinfectants in preventing formation of biofilms on plastic surfaces, higher concentrations of preparations were needed as compared to their bactericidal concentrations for culture of Legionella determined by method of serial dilutions.

[Biofilm formation by Legionella spp. in experiment].

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.

[Electron microscopic study of Burkholderia cepacia biofilms].

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilms) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated.

[Mechanism of interaction of tumor necrosis factor (TNF-alpha) of macroorganism with the Salmonella enterica cells (ser. Typhimurium)].

The goal of this work was to elucidate the mechanism of direct interaction of bacterial cells with tumor necrosis factor (TNF-alpha; cytokine). It was shown earlier that this interaction facilitated activation of bacterial growth and recultivation of non-cultivated forms in vitro and in vivo. It was shown in experiments with mice deficient in the genes encoding eucaryotic TNF-alpha receptors and infected with salmonella that addition of exogenous TNF-alpha to suspension of infection cells caused a one-day acceleration in the infection start (bacteria planting from spleen) in both knockouted and control mice relative to the same animals infected with the same bacteria without cytokine. Thus, bacteria are able to interact with cytokine even in the absence of eucaryotic receptors. Specificity of the bacterium-cytokine interaction and bacterial protein EF-Tu mediating direct interaction of bacteria with cytokine were identified using the method of immobilization of recombinant protein TNF-alpha-spacer-CSD on cellulose.

[Formation of biofilms as an example of the social behavior of bacteria].

This paper is a brief review of data on bacterial biofilms that occur inside and outside of host organisms. Such biofilms are of great ecological and clinical importance. The role of interspecies communications in the development of bacterial biofilms and infectious diseases is particularly emphasized. Considerable attention is given to the electron microscopic study of biofilms formed by Salmonella typhimurium cells incubated as a broth culture in microtubes without aeration. Bacterial samples taken from the biofilm and planktonic culture grown in the same microtube were comparatively investigated by transmission electron microscopy.

[Communication systems in bacteria and their role in pathogenicity].

A review of the literature about the bacterial systems of regulation of expression of the genes controlled by signals generated by the bacteria themselves is given. Three types of presently known Quorum sensing systems contributing to the expression of the pathogenicity factors, infection process, and formation of biofilms by pathogenic bacteria are described. Possible mechanisms of enhanced resistance of bacteria in films to antibacterial preparations and factors of immune protection of human body are discussed. The perspectives of the development of new approaches to treatment of chronic diseases caused by the pathogens producing bacterial films are considered.

[The role of "quorum sensing" regulation system in symbiotic interaction of bacteria Burkholderia cepacia and Pseudomonas aeruginosa during mixed infection].

Data concerning mechanisms of strengthening virulence of bacteria B.cepacia and P.aeruginosa during experimental mixed-infection are submitted. Results of in vitro studies of acylhomoserine lactones (AHL) with various length of a carbon lateral circuit have shown that butanoyl-homoser-ine lactone (C4), hexanoyl-homoserine lactone (C6) and octanoyl-homoserine lactone (C8) separately and in various combinations display different effects on growth of bacteria, formation of colonies and production of pathogenicity factors. Experiments on animals have shown strengthening virulence of bacteria B.cepacia by C4, C6 and C8 lactones when introduced simultaneously. Strengthening of virulence of the P.aeruginosa strain under the action of lactone C6 and simultaneous action of C4, C6 and C8 lactones was observed. The data obtained give grounds for assumption, that bacteria may use exogenous lactones, including those produced by closely related bacteria, during their life cycle.

[Biofilm formation by different strains of Salmonella typhimurium in artificial systems].

The ability of 14 different strains of Salmonella typhimurium to biofilm formation depending on genotype and culture conditions was investigated in artificial systems: in 96-well plastic microtitre plates, plastic and glass tubes, plastic Petri dishes and on microscope glasses. Quantitative biofilm growth was monitored by using an assay based on crystal violet staining, while planctonic growth in the same cultures was monitored by absorbance in iEMS Reader MF, and qualitatively--by digital photo and visually. Optimal rate between growth and biofilm indications for all strains was determined at initial cell concentration 10(6-7) KOE/ml and culture incubation at t degrees 28 degrees C. The nutrient content of the medium significantly influenced the quantity of produced biofilm. The nutrient broth LB without NaCl was more effective in promoting biofilm formation, than LB itself. The least quantity of biofilm was formed in water. The genotype of the strains also critically influenced the quantity of produced biofilm. Nonmotile mutants cells had reduced ability to form biofilm. RpoS mutant cells produced significantly less biofilm as compared with cells of isogenic parent strains. The chemical content of plastic and glass also influenced biofilm formation.

[The role of tumor necrosis factor-alfa in the interaction between the macro- and microorganism].

The authors present a review containing their own and literature data on the role of cytokine TNF-alfa in infectious process on the part of the macro- and microorganism, paying special attention to the latter. The paper contains data from literature concerning the mechanism of the interaction between cytokine and eukaryotic cells and experimental data on direct interaction between cytokine and bacterial cells in vitro and in vivo.