The effect of enzymes carbohydrases exopolysaccharide matrix of biofilms formed by gram-positive and gram-negative bacteria on abiotic surfaces was studied. The ability of a mixture of carbohydrase enzymes (hydrolases and lyases) to completely destroy the matrix of biofilms formed by gram-positive and gram-negative bacteria causing health-associated infections has been confirmed. An optimal mixture of carbohydrases possesed high anti-biofilm activity even when employed in relatively small concentrations and at a brief exposure time.

Задача повышения эффективности антибактериальной ФДТ делает актуальными создание и исследование фотосенсибилизаторов (ФС) на основе поликатионных синтетических бактериохлоринов. Целью настоящей работы было изучить в широком диапазоне концентраций фотофизические и антибактериальные свойства наноструктурированного ФС на основе 3-Py4BСHp4Br4, тетракатионного амфифильного производного синтетического бактериохлорина. Наноструктурированную дисперсию ФС получили путем его солюбилизации в 4%-м Kolliphor ELP (BASF). Исследование интенсивности и формы спектров поглощения и флуоресценции в диапазоне концентраций от 0,001 до 0,2 мМ продемонстрировало низкую агрегацию этого ФС во всем диапазоне и высокую эффективность фотодинамической инактивации грамположительных бактерий S. aureus и грамотрицательных бактерий P. aeruginosa и K. pneumoniae.

New cationic photosensitizer for photodynamic antibacterial chemotherapy based on Chremophor dispersion of using methyl ester 13 3 -N-(N-methylnicotinyl) purpurinimide was developed. It was shown the complete photodynamic inactivation of bacteria Pseudomonas aeruginosa biofilms by using new photosensitizer at the concentration of 1 mM, the incubation time of 1 hour and the irradiation dose of 105 J/cm 2.

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.

С помощью пласпозонного мутагенеза получены мутанты Burkholderia cenocepacia 370 с измененной продукцией N-ацил-гомосеринлактонов (АГЛ) – сигнальных молекул Quorum Sensing системы регуляции. Для определения локализации вставок пласпозонов у мутантных штаммов проведены клонирование фрагментов хромосомной ДНК, содержащих пласпозоны, секвенирование прилежащих к ним участков ДНК и поиск гомологичных нуклеотидных последовательностей в банке генов. Показано, что инсерция пласпозона в ген lon, кодирующий протеиназу Lon, приводит к резкому уменьшению синтеза АГЛ. При инсерции пласпозона в ген pps, кодирующий фосфоенолпируватсинтазу, наблюдается увеличение продукции АГЛ. В мутанте с инактивированным геном lon было отмечено резкое снижение внеклеточной протеазной, гемолитической и хитинолитической активностей по сравнению с исходным штаммом и не наблюдались изменения липазной активности. Мутация в гене pps не влияла на эти свойства B. cenocepacia 370. Мутации в генах lon и pps снижали вирулентность бактерий при заражении мышей.

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.

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.

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.

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.

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.

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.

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.

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.