Background and Aim: The study of biofilm-forming ability of Gram-negative microflora has great practical importance for assessing the effectiveness of antibiotic therapy and finding new ways to diagnose and inhibit the growth of biofilms. This is because poor penetration of antibacterial drugs into the biofilm can lead to the selection of resistant strains and has a consequence evident by the occurrence of relapse of infection in animals. This study aimed to evaluate morphological and densitometric indicators of biofilm formation as well as adhesive properties of Klebsiella pneumoniae. Materials and Methods: K. pneumoniae was cultured at 37°C for 2-144 h in vitro. The specimens for optical microscopy were prepared by fixation with a 1:1 alcohol-ether mixture for 10 min and stained with a 0.5% solution of gentian violet for 2 min, and the optical density index was evaluated at a wavelength of 490 nm. Further, the adhesive properties of the microorganisms were determined at a concentration of 1 billion/ml and a suspension of ram erythrocytes at a concentration of 100 million/ml when cultured at 37°C for 24 h. Blood smears were prepared and stained with 0.5% gentian violet. Results: K. pneumonia cultured at 37°C after 24 h on the meat peptone agar formed large, convex, mucous, and white colonies (d=3.0-6.0 mm). With the growth in the meat and peptone broth, uniform turbidity of the medium was observed. Analyzing the optical density indices (density, D), it was found that K. pneumoniae were good producers of biofilms (D=0.528±0.31). Data for indicators of adhesive properties of K. pneumoniae were as follows: Average adhesion index, 4.56±0.14; adhesion coefficient, 1.07±0.52; and adhesion index, 4.26±0.07. The studied bacteria had high adhesive activity. A direct correlation dependence (R=0.94) of the optical density of biofilms (D≥0.514-0.551) and AAI (4.15±0.28-4.76±0.75) was established. Conclusion: This study has demonstrated that K. pneumoniae had high adhesive activity, was strong producer of biofilms, and the optical density of the sample exceeded the optical density of the control by more than 4 times.

The paper demonstrates morphometric and densitometric parameters of microbial biofilms recovered from lambs with digestive disorders. Changes of quantitative and species composition of the intestinal microbiocenoses in the lambs with digestive disorders were compared with the ones of the clinically healthy lambs. Light microscopy results demonstrated formation of three-dimensional biofilm structure in the form of dense grid consisting of gram-negative and gram-positive bacteria, yeast cells, hyphas and pseudohyphas surrounded with intracellular polymer matrix. Presence of blastospores aided to the increased number of cells attached to the substrate, and biofilm was formed, which consisted of rod and round cells attached to the microfungi cells. In the process of dispersion that occurred during the destruction of the intercellular matrix and bacterial and yeast cell detachment, branched structures separated from the microcolonies and colonized microorganism- free regions of the substrate. The intensity of biofilm formation by the microorganisms under study was evaluated by optic density measurement in 48 hours of cultivation. Fluorescence microscopy results demonstrated that the dynamics of changes of the viable microbial structures was specified by intermittent periods of increased or decreased biofilm formation intensity. Cells characterized by active growth and replication and forming alternating subpopulations were detected in the examined microbial cultures. When determining the viability of the microorganisms in the biofilms, the viable (green fluorescence) and non-viable (red fluorescence) cells were differentiated.

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The article presents the general patterns of formation of a three-dimensional multilayer heterogeneous structure biofilms in vitro, ex vivo, in vivo. It was found, that the mass fraction of cells of biofilms was 86,8-91,4%, of the matrix – 8,9-13,2%, which is due to the structure of the cell wall of microorganisms synthesizing the extracellular matrix. The advantages of transmission and scanning electron microscopy are the visualization of L-shapes and the quantitative assessment of the diameter, perimeter, height, area, volume and chemical composition of cells and the extracellular matrix of microorganisms. Laser modulation interference microscopy made it possible to study biofilms in real time without fixing and staining microorganisms; phase-contrast microscopy – intrapopulation and interpopulation interactions depending on changes in the pH of the environment, oxygen content and other chemical and physical factors. Fluorescent microscopy is a promising method for detection of non-culturable viable cells of microorganisms. The use a modified Gram stain, the use of solution of «Crocus sativus» made it possible to differentiate gram-negative and gram-positive microorganisms and to assess the degree of repair of tissue. The developed new methodological approach made it possible to scientifically substantiate and experimentally confirm the effectiveness of the use of nutrient media containing components for the repair of the cell wall of microorganisms.

The relevance of the study and the presence of gaps in the existing knowledge on the topic. Monitoring studies of the biological safety of food raw materials for microbiological indicators is an urgent problem due to the increase in the number registered diseases transmitted to humans through raw materials and products of animal origin. There is a tendency for a statistically significant increase in epidemiological indicators throughout the world, the proportion of these pathologies is increasing both in humane medicine and veterinary medicine. The aim of the work is a comparative assessment and selection of effective methods for studying the formation biofilms enterobacteriaceae circulating among susceptible animal species and isolated from food raw materials.Methods. Аnalysis of growth and dynamics development biofilms Enterobacteriaceae was carried out during cultivation on nutrient media containing growth factors for the repair of the cell wall and the reversal viable uncultured microorganisms. To study the morphological and functional patterns of the development a population microorganisms in vitro and in vivo, we used the conventional and developed methods for preparing preparations for scanning, transmission phase contrast, optical and luminescence microscopy.Results and its discussion. During microbiological control critical points in the technology of animal husbandry and food production, the morphological and functional characteristics biofilms, which are communities microorganisms secreting a polymer matrix and adhered to the tissues of susceptible animal species and abiotic surfaces livestock buildings and food industries, were studied. The developed methods of biofilm cultivation made it possible to study enterobacteriaceae biofilms in vitro and in vivo, without disturbing the natural architectonics of the population microorganisms, to determine the components extracellular matrix. For the study dynamics morphological and functional patterns of the development populations microorganisms, routine and technological advances present are recognized as promising, for example, scanning electron microscopy makes it possible to assess the degree of formation and morphological composition biofilms. Phase contrast microscopy to reveal processes depending on the composition medium and the oxygen content in the culture medium.Conclusions. Methods for cultivating biofilms in vitro and in vivo without disturbing the natural architectonics of biofilms made it possible to optimize the preparation samples for research and eliminate the routine stages of colony counting, and significantly increase the number of analyzes. Due to the simplicity of operations and minimization manual labor, productivity increases, safety of work is increased, cost of personnel working time is reduced, and subjective factors are excluded. For the development of a complex antiepizootic and diagnostic measures, a priority direction is the disclosure of scientific knowledge in the field fundamental studies of ecological plasticity and adaptation potentially pathogenic enterobacteria to parasitism in the warm-blooded organism of birds and mammals. This will allow solving applied problems controlling the critical points of livestock and food production technology, developing effective chemotherapeutic and disinfecting drugs to reduce cell coaggregation and detect viable uncultured microorganisms.