Effect of Subinhibitory Concentrations of Macrolides on Expression of Flagellin inPseudomonas aeruginosaandProteus mirabilis

Abstract

In the present study we showed by molecular analysis that the inhibition of motility by macrolides in Proteus mirabilis and Pseudomonas aeruginosa was well correlated with the loss of the expression of flagellin. Erythromycin, clarithromycin, and azithromycin at subinhibitory concentrations (sub-MICs) suppressed the expression of flagellin dose dependently. Azithromycin had the strongest inhibitory effect on the expression of P. aeruginosa flagellin, whereas 16-membered rokitamycin had only a weak inhibitory effect. These results indicate the potential effectiveness of sub-MICs of erythromycin, clarithromycin, and azithromycin for the treatment of patients with P. mirabilis and P. aeruginosa infections.Macrolide antibiotics have been widely used to treat various infections. They bind to the 50S ribosomal subunit, resulting in blockage of transpeptidation and/or translocation. The antimicrobial activity of macrolides is broad in spectrum, being exhibited against gram-positive and some gram-negative bacteria, such as Neisseria spp., Campylobacter spp., Haemophilus spp., and Legionella spp. (4). In contrast, most species of Enterobacteriaceae and nonglucose-fermenting gram-negative rods such as Pseudomonas spp. are innately resistant to macrolides (15).Some reports have demonstrated that the treatment of bacteria with subinhibitory concentrations (sub-MICs) of macrolide antibiotics suppressed the expression of bacterial virulence factors in various gram-negative rods (5, 8). Although Pseudomonas aeruginosa is generally highly resistant to macrolides, erythromycin (ERY) suppressed the production of exotoxin A and proteases by this organism at concentrations well below the MIC (6). It was also demonstrated that sub-MICs of macrolide antibiotics decreased protein synthesis (18), enhanced the sensitivity of P. aeruginosa in serum (17), and suppressed biofilm formation through inhibition of alginic acid (7). Based on these reports, certain macrolides at sub-MICs were expected to have clinical effects on patients with respiratory infections caused by gram-negative rods such as P. aeruginosa, and in fact long-term low-dose administration of ERY produced clinical improvement in patients with diffuse pulmonary panbronchiolitis associated with P. aeruginosa infection (16).The present study was therefore undertaken to clarify the in vitro effectiveness of sub-MICs of macrolides as assessed by the inhibition of flagellin expression in P. aeruginosa and Proteus mirabilis. Flagella are among the virulence factors of gramnegative rods and have a role in the initiation of biofilm formation (14). Early studies showed that exposure to sub-MICs of the macrolide azithromycin (AZM) resulted in loss of motility due to the absence of flagella in P. mirabilis and P. aeruginosa (10, 11). However, these observations were based on the conventional light-microscopic examinations and therefore were not quantitative. We describe here the results of molecular analysis of the flagellin inhibition by sub-MICs of macrolides.Nonmucoid P. aeruginosa ...

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