Вплив детергенту, хімотрипсину та фагів на утворення біоплівок стафілококами

Abstract

У статті представлено результати досліджень впливу детергенту (додецилсульфату натрію), хімотрипсину, і стафілококового фагу на утворення біоплівки культурою Staphylococcus aureus. Встановлено, що всі перелічені чинники виявляють відносно стафілококової біоплівки руйнівну дію, ступінь якої корелює з підвищенням концентрації означених речовин; В статье представлены результаты исследований влияния детергента (додецилсульфата натрия), химотрипсина, и стафилококкового фага на образование биоплёнки культурой Staphylococcus aureus. Установлено, что все перечисленные факторы проявляют относительно стафилококковой биоплёнки деструктивное действие, степень которого коррелирует с повышением концентрации указанных веществ; The aim of this work was investigation of influence of sodium dodecyl sulfate, chymotrypsin and phages on formation of biofilms by staphylococcus. Biological objects of study were able to film formation of clinical strain of Staphylococcus aureus isolated from patients with disorders of the gastrointestinal tract and bacteriophages from commercial preparation «Bacteriophage staphylococcal liquid» (JSC «Biopharma», Ukraine). To establish a dedicated clinical strain belonging to the species S. aureus were checked its physiological and biochemical properties, such as hemolytic, lipase and lecithinase activities in accordance seeding bacteria on blood and milk-yolk-salt agar, and also plasmocoagulase activity was determined using citrate rabbit plasma. Staphylococcus are capable to strike almost any organs and tissues of the human body. One of the important factors of pathogenic S. aureus is formation of biofilms, which is part of the life cycle and protect of bacteria from adverse environmental factors. The forming film staphylococcus rapidly acquire antibiotic resistance, and in microbial associations with other agents resistance is even more pronounced. The factors of internal environment such as hormones, enzymes, antibodies, endogenous antibiotics and other metabolites influence essentially on biofilmforming ability of opportunistic microorganisms. The substances whose effects on biofilm studied, chosen not by chance. Thus, there is no doubt that the process of film-forming on the mucosa of the intestine depend on digestive enzymes, especially proteases (trypsin and chymotrypsin) that can break down the protein components in the cell walls and membranes of bacteria and in matrix of biofilms. Determination of detergent effects on film-forming ability is important to develop means of disinfection. The study of bacteriophages action is need for creation of effective drugs for treatment of staphylococcal infections caused by film-forming strains. For biofilms growing 0.4 ml suspension of S. aureus, 1.5 ml nourishing broth and 0.1 ml examined substances in according concentration carried in each well of laboratory plate. The samples were incubating at 37°С during 72 h. After cultivation the medium rest removed from wells, the sediment dissolved in 2 ml physiological solution and tested the optical density at 540 nm. Definition of sodium dodecyl sulfate (SDS) influence on formation of biofilms by staphylococcus shown that optical density of bacteria in biofilms decreases with increasing concentrations of SDS. At the minimal concentration of SDS (0.05 mg/ml) the number of bacteria in the biofilm was 58.7% of control, increasing detergent concentrations (0.5 and 5 mg/ml) some more reduced biofilm density, but not so much as expected, and amounted to 52.3 and 42.2% of controls. Based on the findings, we can suggest that sodium dodecyl sulfate, as detergent, causing destructive effect on the cells of themselves staphylococcus and the elements of the matrix, so that part of bacteria fixed in the biofilm can come in free planktonic phase, and the density of the film as a result significantly reduce. In the study chymotrypsin impact on the biofilm-formation the enzyme concentrations selected so that they would be equivalent to the one that really exist in the body. The chymotrypsin concentration in healthy human intestinal is approximately equal to 0.025% of the total volume of secretions of the slim intestine. So the experimental final concentrations of enzyme in the wells of plate were 0.05, 0.025 and 0.0125 mg/ml. The optical density figures of staphylococcus suspensions in biofilms for increasing concentrations of chymotrypsin (0.0125, 0.025 and 0.05 mg/ml) were reduced after 3-day incubation with the enzyme to 63.3, 51.4 and 36.7% compared to control. Therefore destructive action of chymotrypsin relatively biofilms raise with increasing concentration of the enzyme. To study the influence of phage on the formation of staphylococcus biofilms used commercial preparation Bacteriophagum staphylococcus fluidum. The phage activity is first checked by Gracia method in their ability to form negative colonies on the medium with investigational clinical strain of S. aureus. The initial titre of bacteriophage was equal to 8,0 × 108 BFU/ml (bacteriophage forming units) and its final concentration in the wells was 4,0 × 107 BFU/ ml. The obtained experimental data had demonstrated reducing of staphylococcus biomass in biofilm almost twice after three days incubation with the bacteriophage, namely the optical density of microbial suspensions obtained from biofilm decreased to 51% compared to the control of native biofilm.