Sposobnost formiranja biofilma različitih sojeva Salmonella Enteritidis i inhibitorni efekat etarskih ulja na inicijalnu adheziju i formirani biofilm | Ability of biofilm formation the different strains of Salmonella Enteritidis and inhibitory effect of essential oils on the initial adhesion and preformed biofilm

Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination. Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure. Determination of colony morphotype on Congo red agar at incubation temperature of 25°C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface. Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25ºC. In the case of assays conducted at 37°C, this correlation was not established, except for the isolate SE8. All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25ºC had more favorable effect on the adherence, with the exception of isolate SE3 (p>0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p>0.05). Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was obtained for RDAR than BDAR morphotype isolates (p<0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates. Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h). Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices. Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates. Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum > O. vugare = Th. vulgaris > Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference. Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p>0.05). Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p<0.05).