Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa

Abstract

We demonstrated that a 2% suspension of Pseudomonas aeruginosa alginate completely blocked the diffusion of gentamicin and tobramycin, but not that of carbenicillin, illustrating how alginate production can help protect P. aeruginosa growing within alginate microcolonies in patients with cystic fibrosis (CF) from the effects of aminoglycosides. This aminoglycoside diffusion barrier was degraded with a semipurified preparation of P. aeruginosa alginate lyase, suggesting that this enzyme deserves consideration as an adjunctive agent for CF patients colonized by mucoid strains of P. aeruginosa.

FIG. 1

Effect of alginate on the antibiotic activity of aminoglycosides. The abilities of 200 μg of gentamicin and 100 μg of tobramycin to inhibit the growth of P. aeruginosa FRD2 were examined with either 1% Noble agar (control) or various concentrations of purified P. aeruginosa alginate in the insert cup. Zone sizes determined with 1% Noble agar were considered to be 100% activity, and test zone sizes were compared to these control values. Percentage of inhibition was calculated as follows: 100 × [1 − (radius measured with alginate in the sandwich cup assay/radius with 1% noble agar in the sandwich cup assay)]. Data represent the means ± standard errors of the means based on at least four experiments.

FIG. 2

Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of semipurified alginate lyase. Lane 1, molecular weight standards (in thousands); lane 2, crude enzyme preparation; lane 3, enzyme preparation after fractionation on a Sephacryl H300 column; lane 4, enzyme preparation after fractionation on a Sepharose HP ion-exchange column.

FIG. 3

Effect of AlgL on alginate inhibition of aminoglycoside activity. The abilities of 200 μg of gentamicin and 100 μg of tobramycin to inhibit the growth of P. aeruginosa FRD2 were examined with either 1% Noble agar (control) or 2% FRD1 alginate with various concentrations of AlgL in the insert cup. Zone sizes measured with 1% noble agar were considered to represent 100% antibiotic activity, and zone sizes obtained with 2% FRD1 alginate plus AlgL were compared to these control values. Data represent the means ± standard errors of the means based on at least three experiments.

FIG. 4

Effect of AlgL on alginate inhibition of aminoglycoside penetration. The abilities of 200 μg of gentamicin and 100 μg of tobramycin to inhibit the growth of P. aeruginosa FRD2 were examined with either 1% Noble agar (control) or 2% 144 M alginate with various concentrations of AlgL in the insert cup. Zone sizes measured with 1% Noble agar were considered to represent 100% antibiotic activity, and zone sizes obtained with 2% 144 M alginate plus AlgL were compared to these control values. Data represent the means ± standard errors of the means based on at least three experiments.