Use of confocal microscopy to analyze the penetration of fluorescent complex through Escherichia bacterial biofilms

The study is aimed at analyzing the rate of penetration of the gentamicin antibiotic through biofilms of Escherichia coli and Escherichia albertii bacteria by using confocal laser scanning microscopy (CLSM). The relevance of the study is due to the global problem of antibiotic resistance aggravated by the protective properties of biofilms which reduce the effectiveness of antibiotics 10-1000 times as compared to planktonic cells. The research goal was to evaluate the depth and rate of penetration of the fluorescent complex “gentamicin-acridine orange” into biofilms of E. coli ATCC 25922, E. coli M-17 GKPM 240418 and E. albertii which was first isolated in the Russian Federation. The biofilms were grown for 48-92 hours on cover glasses. The gentamicin-acridine orange complex (gentamicin concentration 2.5 mg cm³) was applied to biofilms for 5, 15, 30 and 60 min at 37°C. The penetration was visualized and measured using CLSM (LSM 800 ZEISS). The penetration depth of the complex increased with exposure time for all strains. The slowest diffusion was observed for E. albertii (15.4 ± 0.5 μm in 5 min; 19.5 ± 0.5 μm in 60 min). Significantly faster penetration was observed for E. coli ATCC 25922 (18.5 ± 0.7 μm in 5 min; 23.5 ± 0.6 μm in 60 min) and especially for E. coli M-17 (15.6 ± 0.3 μm in 5 min; 30.2 ± 0.3 μm in 60 min; p < 0.05). E. albertii required 55 min to reach a depth of 4 μm, while E. coli ATCC 25922 and M-17 reached 5 μm and 15 μm in the same time, respectively.