Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms

Abstract

The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 microm into the biofilms, which averaged 210 microm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band, approximately 30 microm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h(-1). These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

FIG. 1.

Growth of colony biofilms under aerobic conditions (solid circles), anaerobic conditions (empty circles), and anaerobic conditions with nitrate (gray circles). All colony biofilms were grown under aerobic conditions for the first 4 h and were then transferred to alternative conditions at that time, indicated on the graph by the dashed vertical line. The error bars represent the standard error of the mean for each value.

FIG. 2.

Oxygen concentrations in mature (48-h-old) P. aeruginosa colony biofilms (solid circles) versus sterile agar (empty circles). The result shown is the average for three oxygen concentration profiles.

FIG. 3.

Pattern of protein synthesis in P. aeruginosa colony biofilms that were 48 h (A) or 4 h (B) old. Green corresponds to induced GFP and red derives from a counterstain for all biomass. The supporting membrane and agar were at the bottom and the air was at the top in this frozen section of the colony.

FIG. 4.

Effect of oxygen availability on antibiotic susceptibilities of young (4-h-old) P. aeruginosa colony biofilms. The error bars represent the standard errors of the means for each value. •, control; ○, ciprofloxacin; ▵, tobramycin; □, carbenicillin; ▿, ceftazidime; , chloramphenicol; ◊, tetracycline).

FIG. 5.

Effect of nitrate availability on antibiotic susceptibilities of young (4-h-old) P. aeruginosa colony biofilms under aerobic (A) and anaerobic (B) conditions. The error bars represent the standard errors of the means for each value. •, control; ○, ciprofloxacin; ▵, tobramycin; □, carbenicillin; ▿, ceftazidime; , chloramphenicol; ◊, tetracycline.