Keywords
clarithromycin
antibacterial agents
biofilms
Quorum sensing
virulence factors
Pseudomonas aeruginosa
genes expression
Abstract
Second-generation macrolides (azithromycin and clarithromycin) are widely used in respiratory diseases. In addition to antibacterial activity against planktonic cells of gram-positive microorganisms, they have antibiofilm and antivirulent effects, in particular against Pseudomonas aeruginosa. However, information on the genetic regulation of this antivirulent effect is limited and requires comprehensive research. The aim of the study – to evaluate the effect of macrolide antibiotics azithromycin and clarithromycin on virulence factors and activity of genes associated with them in P. aeruginosa. The minimum inhibitory concentration (MIC) of macrolides against the clinical test strain P. aeruginosa 449 was determined by the serial microdilution method. The antivirulent properties of azithromycin and clarithromycin were evaluated in the range of concentrations from 0.15 MIC to 2.0 MIC, determining hemolytic and protease activity, bacterial motility and expression of genes responsible for Quorum sensing-dependent processes. The effect of macrolides (0.5 MIC) on gene expression in P. aeruginosa was studied using quantitative real-time polymerase chain reaction by the 2-ΔΔСt method. The ANOVA method was used for statistical processing of the results. It was established that the macrolides azithromycin and clarithromycin exhibit antivirulent properties against P. aeruginosa. Under the action of azithromycin and clarithromycin, the protease activity of bacteria decreases by 10.1–24.8% compared to the control. The inhibitory effect of azithromycin on the hemolytic activity of P. aeruginosa caused by extracellular hemolysins was recorded at 0.15 MIC and 0.25 MIC (10.1- and 10.4-fold reduction), and on cell-associated hemolysins at 0.15–0.5 MIC (2.9–3.7- fold reduction). The effect of clarithromycin on the hemolytic activity of bacteria is inferior to azithromycin. The effect of macrolides on bacterial motility depends on the type of migration, the drug, and its concentration, but the most pronounced effect was observed on swimming- and swarming-migration. The data obtained indicate that azithromycin and clarithromycin cause changes in the expression of genes that regulate the synthesis of virulence factors. Under their influence, the expression of the aprA, exoA, and toxA genes is significantly reduced. The transcriptional activity of the exoS gene under the action of azithromycin increases (by 13.5 times), clarithromycin does not change. Macrolides reduce the expression of the lasI, rhlI (azithromycin only), rhlR, pqsR (clarithromycin) genes by 2.5–20 times and increase the activity of the lasR. pqsR (azithromycin) genes by 1.9–3.7 times, which are involved in the functioning of Quorum sensing systems in P. aeruginosa. Thus, the antivirulent activity of azithromycin and clarithromycin against P. aeruginosa is realized by influencing the hemolytic, protease activity and motility of bacteria. Macrolide antibiotics, by changing the level of gene expression, are able to disrupt the synthesis of virulence factors and the functioning of Quorum sensing systems, which play a key role at all stages of the development of the infectious process.
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