Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

Abstract

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.

FIG. 1.

Treatment of P. aeruginosa biofilms with EDTA and gentamicin (Gm). Spinning disk reactor biofilms of P. aeruginosa PAO1 were treated for 24 h at 37°C. (Top) EDTA dose response in PBS, pH 7.4 (white), or 20 mM Tris buffer, pH 7.4 (gray). (Middle) PBS with the indicated concentrations of Gm with (gray) or without (white) 50 mM EDTA. (Bottom) Tris with the indicated concentrations of Gm with (gray) or without (white) 50 mM EDTA. Levels of viable bacteria remaining after treatment were determined by plating. Data are means from two disks from each of two spinning reactors. Error bars, standard deviations.

FIG. 2.

Effect of EDTA on P. aeruginosa biofilm structure. GFP-labeled P. aeruginosa biofilms were grown in flow cells for 6 days. Biofilms were grown at room temperature and treated with 50 mM EDTA for 2.5 h. The biofilm matrix and dead cells were counterstained with propidium iodide (30 μM) prior to EDTA treatment. (Left) Biofilm prior to EDTA treatment. (Right) Biofilm after EDTA treatment. The images were acquired by CSLM. (A) Three-dimensional reconstruction. The combined green (GFP) and red (propidium iodide) channels are presented. The squares are 15 μm on each side. (B) Sagittal views of the green channel only. (C) Sagittal view showing the combined red and green channels. Bars, 20 μm. (D) A 0.5-μm slice of the internal region of the biofilm. The combined red and green channels are presented. Bars, 10 μm.

FIG. 3.

EDTA facilitates biofilm detachment and lysis. A GFP-labeled P. aeruginosa biofilm was grown in a flow cell for 6 days and then treated with 50 mM EDTA. The effluent was sampled at the indicated intervals. EDTA was added to the medium reservoir at time zero and reached the flow cell after 45 to 50 min. (Top) CFU and direct counts of the effluent cells. CFU from an untreated control biofilm effluent are shown for comparison. (Bottom) Time lapse microscope images taken at the indicated times during treatment with EDTA. The biofilm matrix was counterstained with 4 μM propidium iodide (red) prior to treatment. The green (GFP), red (propidium iodide), and combined channels are presented. The squares are 60 μm on each side.

FIG. 4.

Role of divalent cations in EDTA-mediated detachment and killing. GFP-labeled P. aeruginosa biofilms grown in flow cells were treated with EDTA saturated with the indicated divalent cations (50 mM). (Top) CFU in the effluent determined by plate counts. (Bottom) Microscope images taken 3 h after EDTA treatment. The biofilm matrix was counterstained with propidium iodide (4 μM) prior to treatment. The squares are 60 μm on each side.

FIG. 5.

Treatment of P. aeruginosa biofilms with EDTA and divalent cations. Spinning disk reactor biofilms of P. aeruginosa PAO1 were treated for 24 h at 37°C. (Top) Total (planktonic plus biofilm) viable counts. (Bottom) Percentage of viable cells present in the biofilm (gray) or the planktonic phase (white). Treatments were PBS alone or PBS plus EDTA (50 mM), EDTA-Ba (50 mM), EDTA-Mg (50 mM), EDTA-Ca (50 mM) or EDTA-Fe (50 mM). The viable cells, which remained attached to the disks after the treatment, were counted as biofilm cells. The viable planktonic cells were the detached cells in suspension in the treated wells. Data are means from two disks from each of two spinning reactors. Error bars, standard deviations.