SPECTROPHOTOMETRIC ANALYSIS OF THE EFFECTS OF ANTISEPTICS AND EN- ZYMES ON THE EXOPOLYMERIC MATRIX OF BACTERIAL BIOFILMS

SPECTROPHOTOMETRIC ANALYSIS OF THE EFFECTS OF ANTISEPTICS AND ENZYMES ON THE EXOPOLYMERIC MATRIX OF BACTERIAL BIOFILMS

Introduction: Currently, the theory of microbial biofilms (BF), or the association of microbial communities has replaced the plankton forms concept of the microbial pathogen of purulent-inflammatory diseases [1] .
Microorganisms in the BF are able to withstand external factors of aggression, including ultraviolet radiation, dehydration, antibiotics, disinfectants and immune protection factors.Many aspects of the functioning of this multilevel system are still not studied.The factors contributing to the destruction of BF are also not completely defined.Thus, there is a need to apply new approaches to the treatment of BF infections. [2]e aim of the study was to assess the effectiveness of the use of different antiseptics and enzymes on the exopolymer matrix of bacterial BF.

Materials and methods:
Using the methods of cultivation and study of microbial BF, the properties of 30 clinical isolates, retrieved from patients suffering from purulent-inflammatory diseases were studied.6 different types of pathogens were identified.Exopolymer matrices were grown from these isolates and working solutions were prepared by the standard method.
As reagents for which spectrophotometric analysis of the efficiency their disruptive action against the exopolymer matrices was carried out on, we used dimethylsulfoxide 25% (DMSO), hydrogen peroxide 3%, chlorhexidine 2% and 0.05%, acetylcysteine.These substances were used in clinically accepted concentrations.We also evaluated the effect of a number of enzymes against BF matrices : proteinase K, type I, hyaluronidase, deoxyribonuclease, trypsin, and alpha-amylase.These enzymes were used as a 0.1% solutions.The reaction mixture consisted of 0.15 ml of one of the above substances and 0.3 ml of a working suspension of the exopolymer BF matrix.
To prepare the working suspension, the unfrozen matrix suspension was diluted with a 0.9% NaCl solution to an optical density of 2.5 units on a multichannel spectrophotometer at a wavelength of 492 nm and then0.15ml of suspension was placed in a well of a 96-well flat-bottom ELISA plate.Further, a 0.1 M solution of phosphate buffer pH 7.4 was added to the suspension until an optical density of 2 units.In 1 ml of the working suspension, 12.2 mg of a dry matrix and 0.1 mg Congo red were contained.As a preservative, sodium azide 2 mg / ml was added to the suspension.
After incubating for 24 hours at 37 °C., the reaction mixture was centrifuged for 10 minutes at 10,000 rpm (7930 g) to precipitate undissolved matrix components.Then 0.15 ml of the supernatant was transferred into the wells of a flat-bottomed plate for ELISA.
Further, the increasing optical density of the supernatant were taken into account on a multichannel spectrophotometer at a wavelength of 492 nm in comparison with negative control samples, where 0.9% NaCl was used instead of the working suspension.
The activity level was estimated in terms of optical density according to the formula: ACT=(0,101 + 11,04*[Е op -Е c ]) 2 , where Е op -the optical density of the supernatant in the working suspension; Е c -the optical density of the supernatant in the control samples.It is important to note that the correlation of the optical density of the solution on Congo red concentration is linear.
To compare the reliability of the difference in the data in different groups, the Mann-Whitney test was used.Statistical processing of the obtained results was carried out using the programs Biostat, Statgraphics Plus, Statistica Version 10.
Results.The 25% solution of DMSO and proteinase K had a significant reliable effect on spontaneous decomposition of the exopolymeric matrix of BF in the control (p <0.05).The most sensitive pathogens to DMSO (89.55, 65.81-101.5, n = 4) and proteinase K (43.96, 27.7-52.76,n = 4) were different strains of S. mutans.It is also worth noting the high activity of proteinase K (28,35, 9,68-49,01, n = 7) on various strains of E. coli.
The lowest activity for DMSO (14.6, 11.75-39, n = 3) was found on different strains of P. aeruginosae.In the case of proteinase K, the effect was not statistically significant (p> 0.05).It is also worth noting the low effectiveness of proteinase K on strains of S. epidermitis (5.05, 3.32-7.2,n = 4).
It was assumed that this effect was achieved due to the multilevel effect of DMSO on BF, increasing their permeability, and affecting many en-zyme systems such as superoxide dismutase, as well as the ability of proteinase K to cleave peptide bonds between amino-acids in proteins of the exopolysaccharide matrix of BF [3] .

Conclusions and prospects for further research:
1.The resistance of the exopolymer matrix of different bacterial biofilms toward various antiseptics and enzymes is interspecific and intraspecific.
2. DMSO and proteinase K were identified as the most active substances, which were used on exopolymer matrices of different bacterial biofilms.
We envisage a chemical analysis of the BP structure in order to determine the regularities of their destruction under the action of various substances.In particular, the study of the polysaccharide spectrum of the components of BP matrices.