Center for Biofilm Engineering
Abstract:
"Contributions of Antibiotic Penetration, Oxygen Limitation, and Low
Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to
Ciprofloxacin and Tobramycin"
03-003 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.
Walters, MC 3rd, F. Roe, A. Bugnicourt, M.J. Franklin, and P.S. Stewart,
"Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic
Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin
and Tobramycin," Antimicrob. Agents Chemother., 47(1):317-323 (2003).
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