Popov N. N., Malanciuc S. G., Mishyna M. M., Lyapunov N. A.

MICROBIOLOGY

Scentific article

Wide distribution of inflammatory processes, including caused by S. aureus, their tendency to in- crease determines the relevance of this problem and provides a social value. It is impossible to solve this problem without the development and implementation of effective treatment. Therefore, this research is devoted to studying of chlorinated antiseptics (cations) action that are widely used in medical practice in combination with anion stabilizer disodium edetate on S. aureus suspension culture, formed biofilm and planktonic cells ability to form the secondary biofilms. Materials and methods. The subject of the study were antiseptic preparations: 0.01 % solution of decametoxin; 0.01 % solution of myramistinum; 0.01 % solution of benzalkonium chloride; 0.01 % solution cetylpyridine chlo- ride with 0.02 % disodium edetate. Suspensions of microorganisms (Staphylococcus aureus ATCC 25923 = NCDC 25923 = F-49) were prepared with a defined concentration of microbial cells using an electronic device Densi-La- Meter (PLIVA-Lachema a. s., Czech Republic) in McFarland scale according to the instructions of application. The optical density of the formed biofilms on the surface of polystyrene plate after incubation for 24 hours was mea- sured. After incubation to daily S. aureus biofilms research antiseptics and nutritional medium were added and mix- ture was incubated at 370C. By comparing the optical density of research and formed control biofilms a conclusion about the level of biofilms destruction was made. Plankton cells from the daily biofilms were inoculated into plate cell together with suspension nutritional medium and incubated in a moist chamber for 24 hours. Then the de- gree of microbial cells aggregation was assessed. A quantitative expression of the degree of biofilm formation and planktonic cells aggregative ability is the value of the optical density on a spectrophotometer «Multiskan EX 355” at 540 nm. The result was determined in units of optical density of biofilm formation in microorganisms. For statistical analysis of the results Exel software for PC and Biostat were used. Results and discussion. During the study of action of antiseptics containing disodium edetatу on S. aureus suspension culture it was found that after inoculation of research antiseptic solutions and incubation overnight at 370C in a moist chamber the optical density of the daily biofilm compared with the optical density S. aureus biofilms without experimental drugs tends to decrease (2,26 ± 0,28 units of optical density) The density of daily biofilms with antiseptics containing 0.02 % disodium edetate was lower than control values of antiseptics without disodium edetate: 0.01 % solution of decametoxin in 2.75 times (0,569 ± 0,03 and 1,569 ± 0,26 units of optical density re- spectively); 0.01 % solution of myramistinum in 1.7 times (0,923 ± 0,06 and 1,536 ± 0,31 units of optical density respectively); 0.01 % solution of benzalkonium chloride in 1.4 times (0,887 ± 0,04 and 0,923 ± 0,06 units of optical density respectively); 0.01 % solution of cetylpyridine chloride in 1.9 times (0,488 ± 0,03 and 0,903 ± 0,07 units of optical density respectively). Study of the ability of S. aureus daily biofilms, formed after exposure to experimental drugs to produce plank- tonic cells with subsequent formation of secondary biofilms found that the density of the formed new biofilms de- creased in comparison to controls (3,507 ± 0,16 units of optical density): when exposed to a solution of decametoxin with 0.02 % disodium edetate at 13.9 times (0,253 ± 0,02 units of optical density); under 0.01 % solution of myra- mistinum with 0.02 % disodium edentate in 3.1 times (1,147 ± 0,15 units of optical density); under 0.01 % solution of benzalkonium chloride with 0.02 % disodium edetate at 15.2 times (0,231 ± 0,02 units of optical density); under cetylpyridine chloride solution with 0.02 % disodium edentate in 9.6 times (0,366 ± 0,04 units of optical density). Analyzing the results of research antiseptics with 0.02 % disodium edetate action on a daily S. aureus biofilm and planktonic cells ability to form secondary biofilm we can state the fact that there is a destruction of primary biofilm (control value – 2,26 ± 0,24 units of optical density): when exposed to a solution of decametoxin with 0.02 % diso- dium edentate in 4 times (0,569 ± 0,06 units of optical density); under 0.01 % solution of myramistinum with 0.02 % disodium edentate in 3. 5 times (0,645 ± 0,05 units of optical density); under 0.01 % solution of benzalkonium chlo- ride with 0.02 % disodium edentate in 6.3 times (0,357 ± 0,04 units of optical density); cetylpyridinie chloride solu- tion with 0.02 % disodium edentate in 2.9 times (0,768 ± 0,08 units of optical density) and inhibiting of the ability of planktonic cells to form secondary biofilm (control value – 3,507 ± 0,42 units of optical density): under action of decametoxin solution with 0.02 % disodium edetate at 28.7 times (0,122 ± 0,01 units of optical density); 0.01 % solution of myramistinum with 0.02 % disodium edetate at 34.4 times (0,102 ± 0,02 units of optical density); 0.01 % solution of benzalkonium chloride with 0.02 % disodium edetate at 32. 5 times (0,108 ± 0,04 units of optical density); cetylpyridine chloride solution with 0.02 % disodium edentate at 24.7 times (0,142 ± 0,02 units of optical density). Conclusions. Thus, research hasrevealed that to prevent formation of biofilms by S. aureus, as well as for in- hibition of formed S. aureus biofilms such antiseptics with 0. 02 % disodium edetate are in appropriate use that to suppress the production of planktonic cells and destroy S. aureus daily biofilm, as 0.01 % solution of myramistinum with 0.02 % disodium edetate; 0.01 % solution of benzalkonium chloride with 0.02 % disodium edetate and 0.01 % solution of cetylpyridine chloride with 0.02 % disodium edentate. This is due to mechanism of antiseptics action on one hand (based on their molecules direct interaction with hydrophobic lipid membranes of microorganisms, which leads to fragmentation and destruction, and the part of the molecule plunging into the hydrophobic region of membrane destroys layer and increases the permeable ability of the membrane to macromolecular substances and changes enzymatic activity of microbial cells, inhibits the enzyme system which leads to inhibition of the activ- ity of microorganisms and their cytolysis), on the other hand – under the chemical interaction between chlorine- containing antiseptics (cations) with disodium edetatate (anion) is formed equivalent amount of hydrogen ions. The concentration of hydrogen ions in the environment acts on the body, either directly or indirectly through effects on ion status and availability of many ions and metabolites and the stability of macromolecules. If the concentration of hydrogen ions from a solution becomes beyond the normal range for this type of microbe, its life is terminated. Thus, the use of antiseptics with disodium edetate prevents formation of biofilms by S. aureus and promotes the destruction of existing biofilms.

antiseptics, disodium edetatate, biofilms, S. aureus

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«Bulletin of problems biology and medicine» Issue 3 part 3 (112), 2014 year, 249-253 pages, index UDK 579. 861. 2:579. 262:615. 28