Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacterial biofilms are abundant in the environment and are involved in several human bacterial infections (reviewed in references
11,
14, and
31). Of medical importance, biofilms can withstand host immune responses (
19-
21) and are much more resistant to antibiotic treatments than their nonattached, individual, free-living (planktonic) counterparts (
28,
36). For these reasons, biofilm infections are persistent, and individuals often show recurring symptoms following antibiotic therapy. One of the best-studied models for biofilm formation is the bacterium
Pseudomonas aeruginosa (reviewed in references
27 and
30), which causes many types of infections, including biofilm-associated chronic lung infections in cystic fibrosis patients, acute ulcerative keratitis in users of extended-wear soft contact lenses, and bacteremia in severe-burn victims.
The metal chelator EDTA has been shown to cause lysis, loss of viability, and increased sensitivity of planktonic
Proteobacteria to a variety of antibacterial agents (reference
13; reviewed in references
25,
29, and
40). This has led to the use of EDTA as a preservative in many products. Little is known about the influence of EDTA on biofilms of
Proteobacteria. Raad et al. (
32,
33) have shown that EDTA combined with minocycline is an effective treatment for microorganisms embedded in biofilms on catheter surfaces. Their studies focused on
Staphylococcus epidermidis,
Staphylococcus aureus, and
Candida albicans; however, they also reported two cases of
P. aeruginosa-infected catheters where the EDTA-minocycline treatment caused a large decrease in the number of viable biofilm cells (
32). Recently, Kite et al. (
23) reported that tetrasodium EDTA could be used to eradicate biofilms on catheters. Ayres et al. (
3) have examined the effects of permeabilizing agents on antibacterial activity against a
P. aeruginosa biofilm grown on a metal disk. Their results further suggest increased anti-
P. aeruginosa biofilm activity for several antibiotics when combined with EDTA (
3).
DISCUSSION
EDTA has a detrimental effect on the outer membrane permeability of free-living planktonic
Proteobacteria (
15,
25,
29,
40). By chelating divalent cations from their binding sites in lipopolysaccharide (LPS), EDTA facilitates the release of a significant proportion of LPS from the cell (
26). Although prolonged treatments with EDTA are lethal, short treatments increase the permeability of the outer membrane to hydrophobic molecules (
25,
29). Thus, there can be synergy between EDTA and other antibacterial agents (
2,
8,
24). In this study we report that EDTA not only kills
P. aeruginosa planktonic cells but also affects
P. aeruginosa biofilms (Fig.
1 and
2).
Exposure of
P. aeruginosa biofilms to EDTA killed
P. aeruginosa cells and triggered detachment of cells from biofilms (Fig.
3 to
5). CSLM revealed that the majority of the cell population affected by the EDTA treatment resides in the inner regions of the mushroom-like structures. This type of killing or detachment pattern has been observed in
P. aeruginosa biofilms exposed to various conditions (
6,
34,
41). We note that sloughing of cells from the outer regions of the biofilms might also contribute to the detachment process. Chen and Stewart (
9) have previously tested the abilities of various chemical treatments to remove mixed
P. aeruginosa-
Klebsiella pneumoniae biofilms. They reported that EDTA treatment (10 mM) resulted in a 49% reduction in cell counts, and they presented some evidence that this was due to dispersal of biofilm bacteria. The authors hypothesized that calcium was important for stabilizing the biofilm matrix (
9). Other studies have also suggested a role for calcium in stabilizing biofilms of bacteria (
18,
22,
39).
To better understand how
P. aeruginosa biofilms are affected by EDTA treatment, we examined the abilities of different divalent cations to block EDTA-induced detachment and killing. Barium addition did not block killing, but the addition of magnesium, calcium, or iron did (Fig.
4 and
5). The relative stability constants of EDTA for the divalent cations may be ranked in ascending order as follows: barium, magnesium, calcium, and iron. Thus, our data support previous conclusions that magnesium can block lysis of planktonic
P. aeruginosa by EDTA (
1,
7). EDTA is thought to chelate stabilizing magnesium ions from the LPS, causing release of LPS from the outer membrane (
5,
26). Magnesium did not completely block EDTA-induced detachment, but the addition of either calcium or iron did (Fig.
4 and
5). Based on previous work, one might have anticipated an involvement of iron and calcium in biofilm maintenance. In
P. aeruginosa, addition of calcium to growth media increased biofilm cohesiveness, resulting in decreased detachment (
38). Turakhia et al. (
39) demonstrated that addition of EGTA (a calcium-specific chelator) to a mixed aerobic sewage sludge biofilm resulted in immediate detachment of cells from the biofilm. We found similar EGTA effects on detachment from the biofilm, but killing was fivefold lower than that found with EDTA (data not shown). Chen and Stewart (
10) have tested the viscosity of a mixed
P. aeruginosa-Klebsiella pneumoniae biofilm suspension following addition of various cations. They report that addition of iron salts significantly increased biofilm viscosity. The authors concluded that electrostatic interactions contribute to biofilm cohesion and that iron cations are potent cross-linkers of the biofilm matrix (
10).
The use of EDTA to treat biofilm-related infections is being evaluated by several groups, with promising results (
23,
32,
33); however, little is known about how EDTA causes increased killing of biofilm cells. The results of this study suggest that the activity of EDTA against biofilm cells is mediated by chelation of several divalent cations that are required to stabilize the biofilm matrix. Future work will be required to determine their specific role in this process. Our results imply that EDTA chelation of magnesium, calcium, and iron can enhance detachment of cells from the biofilm. EDTA also facilitates the killing of biofilm cells by chelating magnesium associated with the LPS. This dispersal process and the increased cell permeability facilitated by EDTA may also explain the enhanced killing observed in combined EDTA and gentamicin treatment (Fig.
1). This combination may have therapeutic utility.