Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin

Abstract

The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 micro g of tobramycin ml(-1)or 1.0 micro g of ciprofloxacin ml(-1). After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.18 for tobramycin and 1.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.

FIG. 1.

Killing of P. aeruginosa by tobramycin (A) or ciprofloxacin (B). Symbols denote planktonic cells (circles), resuspended biofilm cells (triangles), and colony biofilms (squares). Resuspended biofilm cells were dispersed from biofilms immediately prior to antibiotic treatment. Filled symbols indicate antibiotic treatment and open symbols indicate the corresponding untreated controls. Error bars indicate the standard errors of the means. The initial cell densities were 10.4 ± 0.2 log₁₀ CFU per membrane for biofilms, 10.0 ± 0.1 log₁₀ CFU ml⁻¹ for planktonic cells, and 9.9 ± 0.1 log₁₀ CFU ml⁻¹ for resuspended biofilm cells.

FIG. 2.

Killing of P. aeruginosa in colony biofilms (squares) upon prolonged exposure to tobramycin (A) or ciprofloxacin (B). Filled symbols indicate antibiotic treatment and open symbols indicate the untreated controls. Killing of planktonic bacteria (filled circles) is shown for reference. Error bars indicate the standard errors of the means. The initial cell densities were 10.4 ± 0.2 log₁₀ CFU per membrane for biofilms and 10.0 ± 0.1 log₁₀ CFU ml⁻¹ for planktonic cells.

FIG. 3.

Penetration of tobramycin (squares) and ciprofloxacin (circles) through colony biofilms of P. aeruginosa. Filled symbols indicate an experiment with colony biofilm present and open symbols indicate the sterile controls. Relative penetration is the ratio of the antibiotic concentration measured in the disk on the far side of the membrane assembly to the concentration of antibiotic in equilibrium with the agar. Error bars indicate the standard errors of the means.

FIG. 4.

Transmission electron micrographs of P. aeruginosa colony biofilms. The left column is the 12-h untreated control. The middle column shows a biofilm treated with 10 μg of tobramycin ml⁻¹ for 36 h. The right column shows a biofilm treated with 1.0 μg of ciprofloxacin ml⁻¹ for 12 h. As labeled, the rows indicate different locations within the colony.

FIG. 5.

Oxygen concentration profiles in P. aeruginosa colony biofilms. Data for four replicate colonies are shown (filled symbols) along with duplicate profiles in a sterile agar control (open symbols).

FIG. 6.

Visualization of the spatial pattern of protein synthetic activity in P. aeruginosa frozen sections of colony biofilms. Strain FRD1(pAB1) was grown for a total of 52 h with no IPTG present (A), for 48 h without IPTG followed by 4 h with IPTG (B), or for 52 h in the continuous presence of IPTG (C). The exposure time was the same for all three panels.