INFLUENCE OF ANTIBIOTICS ON BIOFILMS OF PSEUDOMONAS AERUGINOSA STRAINS WITH DIFFERENT LEVEL OF CYCLIC DIGUNOSINMONOPHOSPHATE

Aim of the study was to determine the effect of antibiotics on the formation process and mature biofilm of strains of P. aeruginosa with different levels of cyclic diguanosine monophosphate (c-di-GMP). Methods. As test microorganisms, strains P. aeruginosa PA01, P. aeruginosa PA01 Δ wspF (elevated level of c-diGMP) and P. aeruginosa PA01 pJN2133 (reduced level of c-di-GMP) were used. Cultivation was performed in test tubes to determine minimum inhibitory concentrations of antibiotics (MICs) or 96-well flat-bottomed Nuclon plates in LB medium at 37 °C for 24 hours when studying biofilm formation. To assess the effect of the antibiotics on mature biofilm, they were added to the wells one day after the start of the incubation, removing previously plankton cells. The amount of plankton cells was evaluated spectrophotometrically, biofilm mass was determined by the crystalline violet staining method. Results. After preliminary assessment of susceptibility of the strains to a broad range of antibiotics by the disco-diffuse Kirby-Bauer method, streptomycin, ciprofloxacin and cefepime were selected for study, all of the strains tested were susceptible. Minimum inhibitory concentrations of ciprofloxacin and streptomycin were similar for all the strains of P. aeruginosa and were 0.15 and 10 μg/ml, respectively. P. aeruginosa PA01 pJN2133 (MIC 12.5 μg/ml) was more sensitive to cefepime. MIC of this antibiotic for the other two strains was 20 μg/ml. With the addition of antibiotics at the beginning of the cultivation, they effectively reduced number of plankton cells (by 70–90%) and mass of biofilms (by 40–70%). In this case, there was no difference between strains with different content of c-di-GMP. When exposed to mature biofilms, ciprofloxacin suppressed the formation of persistent cells, but reduced the mass of biofilm only in P. aeruginosa PA01 pJN2133. Cefepime and streptomycin reduced the content of persisters only in concentrations of 5 and 10 μg/ml and had no effect on biofilms. Conclusions. The decreased content of c-di-GMP in P. aeruginosa cells increases the sensitivity of biofilms to antimicrobial drugs. Ciprofloxacin is the most promising antibiotic for the development of combined agents that can prevent the formation of biofilms and destroy them.