The influence of azithromycin on biofilm formation by Pseudomonas aeruginosa, a cause of refractory chronic respiratory tract infection, was investigated. Alginic acid produced by a mucoid strain of P. aeruginosa was quantified by high-performance liquid chromatography from colonies growing on an agar medium. Polysaccharides in the biofilm formed on silicon chips by a nonmucoid strain were determined by a tryptophan reaction. The effect of azithromycin was examined at concentrations below the minimum inhibitory concentration (sub-MIC) for each strain. Azithromycin significantly inhibited the production of alginic acid from the mucoid strain at ≥ 1/256 MIC, and the production of exopolysaccharides from the nonmucoid strain at ≥ 1/16 MIC. The inhibition of biofilm formation by azithromycin was also observed by scanning electron microscopy. These findings suggest that azithromycin inhibits biofilm formation by P. aeruginosa at concentrations well below the MIC.

The in-vitro effects of ten antimicrobial agents on the biofilm formation of Pseudomonas aeruginosa were investigated. The production of alginic acid by mucoid P. aeruginosa cells cultured in agar media with sub-MICs of antimicrobial agents was quantified by high-performance liquid chromatography. Alginic acid production was inhibited by 1/4 MIC of minocycline (P < 0.002) and tobramycin (P < 0.02), and by 1/256-1/1/64 MIC of macrolides (erythromycin, clarithromycin, roxithromycin, and rokitamycin) and clindamycin (P < 0.02), compared with drug-free controls. Piperacillin, ceftazidime, and ofloxacin did not inhibit alginic acid production. The production of exopolysaccharide by non-mucoid P. aeruginosa cells grown on silicone plates in sub-MICs of antimicrobial agents was determined by quantitative tryptophan assay. Exopolysaccharide production was inhibited by 1/16 MIC of macrolides and clindamycin, but not by other antimicrobial agents. Electron microscopy showed that biofilm formation by mucoid and non-mucoid type P. aeruginosa strains was inhibited by sub-MICs of erythromycin and correlated with the in-vitro production of alginic acid and exopolysaccharide. These results suggest that sub-MICs of macrolides and clindamycin suppress biofilm formation by P. aeruginosa and that intractable chronic respiratory tract infections due to P. aeruginosa might be prevented.

The in vitro effect of macrolides at concentrations below the minimum inhibitory concentration (sub-MIC) on the interaction between Pseudomonas aeruginosa biofilm and human polymorphonuclear leukocytes (PMNs) was investigated by using a chemiluminescence assay. The PMN response to either mucoid or nonmucoid P. aeruginosa biofilm was strongly reduced compared with the response to planktonic bacteria (p < 0.01, p < 0.001, respectively). When biofilms were treated with erythromycin, clarithromycin, roxithromycin and azithromycin prior to incubation with PMNs, the chemiluminescence response was markedly enhanced as compared to untreated controls, and a dose-dependent effect was noted over the range of sub-MIC concentrations studied. In general, macrolides appeared to be slightly more active against mucoid biofilm. Azithromycin was shown to be the most active macrolide against P. aeruginosa biofilms. However, the treatment with sub-MICs of rokitamycin did not have any effect. On the other hand, treatment of planktonic bacteria with macrolides before being exposed to the PMNs did not affect the chemiluminescence response as compared to untreated controls. These findings suggest that macrolides inhibiting the biofilm formation of P. aeruginosa may facilitate the phagocytosis of bacteria by PMNs.

Suppression of endotoxin release and subsequent production of inflammatory cytokines is crucial in the treatment of septic shock. We investigated the effect of clindamycin (CLI) on endotoxic shock induced in mice by Escherichia coli lipopolysaccharide (LPS). Mice were treated with CLI (160 to 600 mg/kg) or saline and then injected with E. coli LPS and D-(؉)-galactosamine intraperitoneally 0.5 h after CLI administration. Pretreatment with CLI significantly improved survival in a dose-dependent manner (CLI, at 160, 300, and 440 mg/kg) and significantly lowered the peak concentrations of tumor necrosis factor alpha and interleukin-1␤ (IL-1␤) in serum. However, the peak concentrations of IL-6 in the sera of CLI-treated mice were higher than in control mice. Our findings suggest that CLI alters LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model.

The effect of subminimal inhibitory concentrations (sub-MICs) of antimicrobial agents on the adherence of Pseudomonas aeruginosa (pili+) to trachea mucosal cells, the first stage in the development of P. aeruginosa respiratory tract infections, was investigated. The adherence of P. aeruginosa to the cells of the lower respiratory tract using a model of acid-injured trachea in mice was observed by electron microscopy (transmission and scanning). When P. aeruginosa was cultured with 1/4 MIC of erythromycin, minocycline, clindamycin, ofloxacin or tobramycin at 37 degrees C for 4 hours, the number of pili was significantly reduced (P < 0.01), together with a significant reduction in the number of adherent bacteria at 1/4 MIC of erythromycin (P < 0.01). No suppressive effects of piperacillin or ceftazidime were obtained on the piliation and adhesion of P. aeruginosa. These findings indicate that sub-MICs of erythromycin reduce the adherence of P. aeruginosa to the tracheal mucosa, which may prevent the onset of P. aeruginosa respiratory tract infection.