Study of enterococci biofilm formation using photometric method with biofilm modeling on abiotic polystyrene surfaces

Abstract

Introduction. Static photometric method is widely used to determine the ability of biofilm formation. The essence of this method is to measure the optical density of the dye, eluated with a solvent from the biofilm. The aim of the study was to conduct a comparative analysis of the results of determining the enterococci biofilm formation using photometric method with biofilm modeling in 96-well microtiter plates and 4-section Petri dishes. Material and methods. The study was conducted using 61 strains of microorganisms genus Enterococcus, taken from the clinical material of patients with purulent-inflammatory infections. To perform the study of biofilm formation using 96-well microtiter plates technique, 100 μl of TSB with 1% glucose and 10 μl of enterococci daily culture suspension with density of 0.5 units by Mc Farland, prepared using the Densi-La-Meter II device, were placed in each well. The plates were incubated at 37 °C for 48 hours, washed three times in phosphate-buffered saline (pH 7.2-7.4) of 200 μl volume, dried at 37 °C for 15 minutes. The biofilms were stained with crystal violet 1% aqueous solution, washed with distilled water. 100 μl of ethanol/isopropanol mixture (1:1) were added to the wells. The optical density of wells content was measured with the AIF-C-01S reader at a wavelength of 570 nm and 620 nm. The results were analyzed using Stepanovic S. et al. recommendations, according to which enterococci were divided into strains with high, medium and low degree of biofilm formation. To study the enterococci ability of biofilm formation in 4-section Petri dishes, 1.75 ml of TSB with 1% glucose and 0.25 ml of enterococci microbial suspension with density of 0.5 units by Mc Farland were added in each sector, 0.25 ml of medium were added to the control sector. The dishes were incubated at 37 °C for 48 hours, washed three times in phosphate-buffered saline (pH 7.2-7.4), dried at room temperature for 30 minutes. The biofilms were stained with crystal violet 1% aqueous solution. The dye eluation from the biofilm was performed twice with 2 ml of ethanol/isopropanol mixture (1:1) for 20 minutes. The optical density of eluates was measured with the SF-56L spectrophotometer at a wavelength of 590 nm. Results and discussion. Analysis of the results of the study on enterococci ability to form biofilms with biofilm modeling in 96-well polystyrene microplates revealed that, when using light filters with a wavelength of 570 nm, optical density rate ranged from 0.001 to 1.256 OD₅₇₀ with an average value of (0.264±0.04) OD₅₇₀. It was found that the number of strains capable of biofilm formation amounted to (68.8 ± 5.9)% of all strains used in the study. Enterococci distribution by intensity of biofilm formation was as follows: number of strains with high degree equaled (90.4±4.5)%, share of strains with medium and low degree was significantly lower – each amounted to (4.8±3.3)% (р<0.05). When measuring the optical density of eluates using the AIF-C-01S reader with light filter at a wavelength of 620 nm, it was found that the indicators ranged from 0.001 to 0.436 OD₆₂₀ with an average value of (0.077±0.01) OD₆₂₀. While analyzing the results with consideration of the above criteria, it was found that the share of strains capable of biofilm formation equaled (60.7±6.3)%. The number of strains with high degree of biofilm formation amounted to (75.6±7.1)%, with average and low degree – (13.5±5.6)% and (10.9±5.1)% respectively. The performed statistical analysis showed no significant difference between the number of strains capable of biofilm formation and intensity of biofilm formation when applying light filters with a wavelength of 570 nm and 620 nm (p>0.05). When modeling the enterococci biofilms in 4-section Petri dishes, it was found that the eluates optical density rates ranged from 0.161 OD₅₉₀ to 3.294 OD₅₉₀with an average value of (1.387±0.14) OD₅₉₀. Analysis of the results showed that the number of enterococci strains capable of biofilm formation equaled (62.3±6.2)% and did not differ significantly from the corresponding indicator in the study with 96-well polystyrene microplates (p>0.05). Conclusion. Static photometric method is widely used to determine the enterococci ability of biofilm formation due to its convenience, high performance, and clearness. Microbial biofilms are mostly modeled in 96-well polystyrene microtiter plates. Our study showed the feasibility of also using 4-section polystyrene Petri dishes for enterococci biofilm modeling. It was confirmed that it is necessary to use light filters with a wavelength range from 570 nm to 620 nm when measuring the optical density of the enterococci eluates with a reader, if using the crystal violet dye.