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 g of tobramycin ml ؊1 or 1.0 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 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.

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp 1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 m wide in colony biofilms and 30 m wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 m into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.Biofilms are communities of microorganisms that are associated with surfaces. Cell clusters in biofilms are known to be characterized by gradients in the concentrations of oxygen, nutrients, and metabolic waste products. It is widely recognized that such chemical heterogeneity in microbial biofilms can lead to microorganisms in the biofilm exhibiting different rates of growth and metabolic activity. Even pure cultures of microorganisms growing in biofilms experience these gradients and may exist in a range of metabolic states. The variety of growth states that can be represented in a biofilm, even for a single species, surely contributes to the special ecology and antimicrobial tolerance manifested by biofilms. Given the fundamental significance of growth status, it is surprising that there have been few investigations in which the growth patterns in biofilms have been visualized.Several studies have investigated spatial patterns of cellular activity inside biofilms, using approaches such as staining with nucleic acid dyes that differentially indicate DNA and RNA (17), hybridization to 16S rRNA with fluorescently tagged oligonucleotide probes (9), the induction of alkaline phosphatase followed by staining with a fluorogenic phosphatase substrate (8,19), and green fluorescent protein (GFP) expression from a growth-rate-dependent promoter (11). These previous investigations have revealed gradients in metabolic activity in biofilms.The purpose ...