Inhibition of tobramycin diffusion by binding to alginate

Abstract

[3H]tobramycin bound to sodium alginate and to exopolysaccharide prepared from two mucoid strains of Pseudomonas aeruginosa. Binding to sodium alginate was similar to binding to exopolysaccharide, both in the dependence on tobramycin concentration and in the maximum binding observed at saturation. Incorporation of sodium alginate into agar plates reduced the zone sizes of growth inhibition caused by tobramycin. The reductions in zone sizes were quantitatively accounted for by the binding of tobramycin to sodium alginate during diffusion of the antibiotic away from the well in which it had been placed at the start of the experiment. However, the binding of tobramycin to the exopolysaccharide of P. aeruginosa, and the resulting inhibition of diffusion of the antibiotic, did not significantly increase the penetration time of a spherical microcolony with a radius of 125 ,um, such as might be found in the respiratory tract of a patient with cystic fibrosis (from a 90% penetration time of 12 s in the absence of exopolysaccharide to one of 35 s with an exopolysaccharide concentration of 1.0% [wt/voll).The question of whether bacterial exopolysaccharides reduce the penetration of antibiotics to their target sites (5, 22) is an important one in antibacterial chemotherapy. This is because exopolysaccharide-producing bacteria existing as biofilms are less susceptible to antibiotics than are freely suspended bacteria (6, 17), and mucoid Pseudomonas aeruginosa apparently forms microcolonies (12) when causing respiratory tract infections that are refractory to chemotherapy in patients with cystic fibrosis. Moreover, in a recent review (4), the exopolysaccharide material of the biofilm was specifically postulated to exclude antibacterial substances.Inhibition of the diffusion of aminoglycoside antibiotics occurs in the presence of alginate (21), a polyanionic polysaccharide similar in structure to the exopolysaccharide of mucoid P. aeruginosa (13), or in the presence of the exopolysaccharide from P. aeruginosa (21). A likely reason for the reduced rate of diffusion of aminoglycosides within the anionic polysaccharide matrix is that any antibiotic-binding sites act as sinks, thereby reducing the free concentration of antibiotic, which is effectively the driving force of diffusion. In apparent conflict with this suggestion, the binding of tobramycin and streptomycin to alginate has been reported (23) as not being detectable in a physiological buffer containing 0.10 M NaCl.We quantitatively investigated the inhibition of diffusion and assessed the binding of tobramycin to alginate and Pseudomonas exopolysaccharide using radiolabeled tobramycin. Here we report that in a physiological buffer solution, tobramycin binds to alginate and to exopolysaccharides isolated from two mucoid strains of P. aeruginosa and that the binding to alginate quantitatively accounts for the inhibition of diffusion reported previously (21). However, binding and consequent inhibition of diffusion cannot account for antibiotic resistance within microcolonie...

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