The Theory of Antibiotic Diffusion Zones

[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...

show abstract

“…The principles of assessing diffusion phenomena from the zone sizes of bacterial growth inhibition have been reviewed by Cooper (2,3).…”

Section: Methodsmentioning

confidence: 99%

“…Squared radii of zones of inhibition (r2) were plotted against lnCo for a series of plates that were preincubated at 37°C for various periods of time before the addition of tobramycin (Fig. 2a) S, = 4D(to -h) (3) where h is the preincubation time. Rearranging equation 3 yields h = to -S114D.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of tobramycin diffusion by binding to alginate

Nichols

Dorrington

Slack

et al. 1988

Antimicrob Agents Chemother

275

157

View full text Add to dashboard Cite

show abstract

“…Culture filtrates, harvested after suction filtration of the cultivation broth through linen cloth, were centrifuged (10 000 x g, 15 min), and the clear supernatants were used for the routine assay of penicillin with Sarcina lutea as a test strain (Cooper 1972). Calculations were made according to a calibration curve with penicillin G. The lowest concentration level detectable was 70 pg/L ( * 10%).…”

Section: Analysis Of Penicillinmentioning

confidence: 99%

Regulation of α-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from α-aminoadipate into penicillin biosynthesis

Mach²,

Affenzeller³

et al. 1992

Can. J. Microbiol.

View full text Add to dashboard Cite

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.

show abstract

“…Culture broths were centrifuged at 1800 g for 10 min. The supernatant fluid was centrifuged again for 5 min at 1500 g and the clear supernatants were used for the routine assay of penicillin with Bacillus subtilis ATCC 6633 as test strain (Cooper, 1972). Penicillin was sometimes also determined colorirnetrically with the hy droxylamine reagent (Bcxer & Everett, 1949) to confirm the results.…”

Section: Methodsmentioning

confidence: 99%

Lysine Regulation of Penicillin Biosynthesis in Low-producing and Industrial Strains of Penicillium chrysogenum

Luengo

Revilla

Villanueva

et al. 1979

Journal of General Microbiology

View full text Add to dashboard Cite

The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (AS-P-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.

show abstract

The Theory of Antibiotic Diffusion Zones

Cited by 34 publications

References 26 publications

Inhibition of tobramycin diffusion by binding to alginate

Inhibition of tobramycin diffusion by binding to alginate

Regulation of α-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from α-aminoadipate into penicillin biosynthesis

Lysine Regulation of Penicillin Biosynthesis in Low-producing and Industrial Strains of Penicillium chrysogenum

Contact Info

Product

Resources

About