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10Application of a 1% solution of dihydroquercetin for treatment of the surface of the Petri dishes was significantly delayed the process of adhesion. Within 3 hours was recorded not only delay the adhesion, but the death of microbial cells. While the beginning of the formation of biofilm microbial culture also slowed in the two and a half times.
Keywords. Adhesion, bacteria, biofilm, Escherichia coli, dihydroquercetin
In the diagnosis and prevention of infectious diseases is important to the understanding of the etiological and pathogenetic aspects. Adhesion is the starting point of the infection process [2]. Specific adhesion is the result of molecular interactions between the receptor cells and microbial adhesion. The adhesins of the majority of gram-negative microorganisms enter the liposaccharide component of the pili and the fili, which is why they are called fimbrial adhesins.
In gram-positive bacteria adhesion is carried out by means fimbrial adhesins. Most of them are proteins of the cytoplasmic membrane and teihoic acids of the cell wall. Receptors for adhesins of gram-positive bacteria are most often fibronectin and proteins of the extracellular matrix. Fimbrial adhesins more effective than afimbrial ones [7].
Adhesion in the macroorganism triggers a cascade of complex reactions mediated by iron molecules. Strains of pathogenic E.coli can carry two types of villi: P villi and villi type I. P villas adhesives (PapG protein) bind to the Gala (1-4) carbohydrate fragments of Gal epithelial cell membranes, and type I villi bind to mannose residues [8]. The concentration of iron ions, or rather its deficiency, for the E.coli serves as a marker of the ability to proliferate. The subsequent production of cytokine epithelium by the feedback signal influences the expression of siderophobes and iron binding proteins by the E.coli [4]. Binding of mannose receptors triggers apoptosis, which causes intense exfoliation of epithelial cells. But the apparent elimination of microorganisms does not occur. Because of the exposure of the deep layers of the epithelium to the microorganisms, the underlying cells that do not possess protective functions and are susceptible to invasion become accessible. Similar processes are described in the adhesion of entero-pathogens to the epithelium of the gastrointestinal mucosa, and the Neisseria to the mucosa of the genital tract.
When the microorganism contacts the surface of non-biological materials, adhesion also occurs, but the main stages unfold according to another scheme. First, separate microcolonies are formed on the surfaces, and then expand into a continuous biofilm. Biofilm is a community of microorganisms, covered with a common glycocalysis — a complex polysaccharide polymer. The formation of a biofilm is not a purely mechanical fusion of microcolonies [9]. This process is subject to complex regulation, the leading role in which play autoinducers. The phenomenon of autoinduction, or cooperative sensitivity (quorum sensing). The vast majority of cells are at rest and are characterized by extremely low sensitivity to the effects of antibacterial agents. In some areas of the biofilm, breeding centers periodically occur, resulting in the release of free (plankton) cells of microorganisms into the environment.
In recent years, the influence of various substances on the course of biofilm formation has been studied. It is known, for example, that the processes of adhesion and formation of biofilms vary depending on the content of ions of calcium, magnesium, zinc and manganese in a dose-dependent manner [4]. In addition to inorganic substances, organic compounds also have an effect on microbial adhesion [5]. Biofilms can be created from a population that has evolved from a single species, or from a community that has occurred from numerous microbial species. Often biofilms are formed on the surface of medical devices, catheters, implants and are the cause of the development of infectious complications, often threatening the life of the patient. At least 65% of hospital infections are associated with biofilms [1]. And the bacteria of the family Enterobacteriaceae in aggregate are the most frequent pathogens of nosocomial infections [6]. Prevention of the development of biofilm infections and their control are topical areas of modern medicine.In this connection, we set the goal to study the rate of biofilm formation by the culture of Gram-negative bacteria with different adhesive activity on the example of Escherichia coli with and without the use of the antioxidant dihydroquercetin flavonoid and without it.
Materials and methods. For the study used wild strains of Esherichia coli isolated from feces of healthy people. The study was conducted by microbiological methods. Isolated cultures were incubated in the meat-peptone broth. Daily microbial culture was adjusted to a concentration of 107 CFU and brought to the surface of a plastic Petri dish with a diameter of 40 mm, pre-treated in 1% solution of dihydroquercetin and dried under ultraviolet irradiation. As a control used the cups with the microbial culture of the same concentration, but without treatment with dihydroquercetin.
Results and discuss. Adhesive activity of strains of Escherichia coli were studied by application of 3% suspension of erythrocytes 0(I) group and determining the average adhesion (DAA). The intensity of biofilm formation was evaluated microscopically after 30 minutes, 1 - 1,5 - 2 - 2,5 - 3 hours and 6 hours of incubation of culture. The color was produced by gentian-violet.
In Escherichia coli with a moderate adhesive activity, the DAA was 3.6 microbe cell / Er. In E.coli with low adhesive activity, the DAA was 1.9 microbe cell / Er. In the course of the study, a direct relationship between the rate of biofilm formation and the adhesive activity of microorganisms was found. Thus, in E.coli with medium adhesive activity, the onset of biofilm formation was observed after 2.5-3 hours of incubation. In strains with low adhesive activity, this process lagged behind and was observed only after 6 hours of incubation. The use of dihydroquercetin for the treatment of Petri dishes significantly delayed the adhesion process. Within 3 hours, not only delayed adhesion, but also death of microbial cells was recorded. What can be explained by both natural causes (elapsed life time of a cell individual) and by a harmful change in the osmotic pressure of the medium during the transition of dihydroquercetin into the soluble phase, that is, the known antibacterial properties of flavonoids [3]. Surface treatment with dihydroquercetin slowed the start of biofilm formation by a microbial culture almost two and a half times.
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Амурский медицинский журнал №3 (19) 2017
