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NDT Advance Access originally published online on April 20, 2006
Nephrology Dialysis Transplantation 2006 21(8):2247-2255; doi:10.1093/ndt/gfl170
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Dialysis and Transplantation

Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces

Robert M. Q. Shanks1, Jennifer L. Sargent1, Raquel M. Martinez1, Martha L. Graber2 and George A. O'Toole1

1 Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755 and 2 Department of Medicine, Section of Hypertension and Nephrology, Dartmouth Hitchcock Medical Center, Lebanon, NH 03766, USA

Correspondence and offprint requests to: George A. O'Toole. Email: georgeo{at}dartmouth.edu



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Microbial biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus biofilm formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro biofilm formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS).

Methods. Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on biofilm formation on polystyrene, polyurethane and silicon elastomer.

Results. Sodium citrate at concentrations above 0.5% efficiently inhibits biofilm formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate biofilm formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of biofilm formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated biofilm formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus biofilm formation.

Conclusions. This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the formation of S. aureus biofilms, suggesting that they may reduce the risk of biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates biofilm formation by S. aureus.

Keywords: adherence; anticoagulant; bacteria; biofilm; catheter lock



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Bacterial biofilms are complex microbial communities which have a significant impact upon human health [1–3]. Bacteria in biofilms may have enhanced pathogenic capability relative to bacteria in solution by virtue of sessile behaviour, increased resistance to antimicrobial agents and the potential for detachment and distal embolization of biofilm fragments [4]. There are few clinical situations where this is more relevant than infections associated with indwelling intravenous catheters for dialysis and other purposes. It is estimated that there are over 400 000 vascular catheter-related bloodstream infections each year in the US [5]. Each such episode is expensive to treat and potentially complicated by systemic sepsis and other foci of infection.

Bacterial biofilms are defined as communities of bacteria attached to surfaces [6]. Biofilms defy most antimicrobial treatments and represent a nidus for chronic infections. In a previous study, we found that sodium heparin, which is the most widely used catheter lock solution, stimulates Staphylococcus aureus biofilm formation in vitro [7]. Catheter lock solutions are placed in the catheter lumen between dialysis sessions to maintain catheter patency. Heparin is widely used in the US for this purpose. Heparin is effective, but has several disadvantages. It is relatively expensive and of increasing concern in the dialysis setting in the development of heparin-associated and initiated thrombocytopenia and thrombosis (HIT/HAT) mediated by antiheparin antibodies. Our previous work has shown that heparin, at clinically relevant concentrations, enhances S. aureus biofilm formation. Several previous studies have suggested that solutions of sodium citrate, ethylene diamine tetra-acetic acid (EDTA) and sodium citrate with gentamicin as catheter lock solutions are as efficacious as heparin in preventing thrombosis and may be superior for maintaining catheter patency [8,9]. A recent randomised control trial demonstrates that 30% trichlorectic acid reduced the risk of cather-related sepsis in dialysis patients by 75% when compared with heparin as a lock solution [9]. We have explored the effect of alternative catheter lock solutions upon S. aureus biofilm formation.

Sodium citrate is a potent antimicrobial agent and is effective as an anticoagulant catheter lock solution [10]. Anticoagulant concentrations of sodium citrate ranging from 0.3 to 47% have been described [10]. We found that sodium citrate in this range effectively prevents the formation of staphylococcal biofilms. The effect of sodium EDTA, lepirudin (Refludan), tissue plasminogen activator (Alteplase) and low molecular weight heparin (LMWH; Lovenox) on S. aureus biofilm formation was also assessed.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Strains and medium
Staphylococcal strains used in this study are listed in Table 1. Bacterial cells were grown in tryptic soy broth (TSB) or on tryptic soy agar (TSA). For all phenotypic assays, we used 66% TSB + 0.2% glucose, as this medium promotes robust biofilm formation (data not shown, 20g/l of Difco Bacto Tryptic Soy Broth, Becton Dickinson, Sparks, MD). Henceforth in this article ‘TSB’ will signify 66% TSB.


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  		Table 1. Citrate alters staphylococcal biofilm potential

 
Biofilm studies
Biofilm formation on polystyrene was performed similar to the previously described methods [7]. Overnight cultures of staphylococci were grown in TSB. Cultures were diluted to OD600 of 0.01 in TSB + glucose (0.2%) with experimental solutions added at 10% vol/vol to final concentrations indicated for each experiment in the respective figures. One hundred microlitres of cells plus medium, plus or minus tested substance, were added to individual wells of a tissue culture-treated, 96-well polystyrene microtitre dishes (Costar 3595, Corning Inc., Corning, NY). These were incubated in a closed, humidified plastic container for 8 h at 37°C, then assayed for biofilm formation. Non-adherent cells were removed and adherent cells were stained with 0.1% crystal violet as previously described [7]. Photographs of the inverted plate were taken with a digital camera (Nikon 990, Nikon, Mellvile, NY), then crystal violet was solubilized using 30% glacial acetic acid for 15 min. Relative biofilm formation was assayed by reading optical density at 550 nm using a Molecular Devices Vmax kinetic microplate reader (Sunnyvale, CA). Six or seven wells were used for each dilution, and each experiment was done at least three times, except when testing the group of strains listed in Table 1, when experiments were done at least two times.

Reagents specifically tested in these experiments were sodium citrate (S279–500, Fisher Scientific, Fair Lawn, NJ), lepirudin (Refludan NDC 50419-150-01 Berlex Laboratories, Wayne, NJ), tissue plasminogen activator (Alteplase or Cathflo Activase, NDC 50242-041-65, Genentech, Inc., South San Francisco, CA), LMWH (Lovenox NDC 0075-0624-30, Aventis Pharmaceuticals, Bridgewater, NJ), EDTA (10378-23-1, Fisher Scientific, Pittsburgh, PA) and gentamicin sulfate (G-3632, Sigma-Aldrich Co., St Louis, MO).

Adherence to silicone elastomer and polyurethane
A 1 mm thick sheet of silicone elastomer was acquired from Goodfellow Cambridge Limited (LS269875, Huntingdon, UK). Coupons (1/8'' diameter) were made using a hole punch (FSK-2351, Fiskars Inc., Madison, WI) and three of these coupons were glued to the bottom of a well in a 24-well costar dish with silicone sealant (super silicone sealant 08661, 3M Inc., St Paul, MN). TSB + 0.2% glucose + bacterial cells were added as with the microtitre dish assay, except that 1.0 ml was added per well. Bacterial cells were incubated with the coupons for 24 h, then were washed five times with sterile PBS, stained with crystal violet, washed three times with sterile PBS and allowed to dry overnight. Individual crystal violet stained coupons were then moved to individual wells of a 96-well plate containing 0.125 ml of 30% glacial acetic acid for 15 min. An aliquot of 0.1 ml of solubilized crystal violet were then moved to 96-well dishes and A550 readings were determined as noted previously.

Adherence to polyurethane was determined just as earlier except using coupons punched from a 1.6 mm thick polyurethane sheet (Precision Urethane and Machine, Hempstead, TX).

Microscopy
Microscopy was performed as described previously [7]. Cell viability of adherent bacterial cells was determined with the Live/Dead BacLight Bacterial viability kit (L7012, Molecular Probes, Eugene, OR) following the manufacturers’ directions.

Ion concentration determination
Free calcium and sodium levels were determined using fluorescent ion detection kits from Molecular Probes (Eugene, OR) as prescribed by the manufacturer. Calcium was detected with Fluo-5F and sodium levels with CoroNa (C36675 [GenBank] ). Fluorescence was determined with a Spectramax M2 microplate reader (Molecular Devices, Sunnyvale, CA).

Statistical analysis
Error bars are shown as one-standard deviation. P-values were determined using Student's t-test with Excel software.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Sodium citrate concentrations ≥0.5% prevent S. aureus biofilm formation, but do not destroy existing biofilms
Staphylococcus aureus biofilm formation on polystyrene was assayed with the addition of sodium citrate over a concentration range of 0–4% (Figure 1A). We found that biofilms were stimulated at low concentrations of sodium citrate particularly at 0.125 and 0.25% (Figures 1A–C and 2C–D). At higher concentrations, biofilm formation was largely prevented (Figures 1A–C and 2E–F).


Figure 1
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  		Fig. 1. Sodium citrate inhibits biofilm formation above 0.5%, but strongly stimulates biofilm formation at lower concentrations. (A) Biofilm formation of S. aureus strain MZ100 on polystyrene microtitre plates at 8 h in response to increasing concentrations of sodium citrate. (B and C) Biofilm formation was determined on silicon elastomer coupons as a function of citrate concentrations. Three coupons were attached to the bottom of each well of a 24-well polystyrene dish. At 20 h non-adherent bacterial cells were removed and biofilms were stained with crystal violet. (B) A digital photograph shows biofilm formation on polystyrene and silicon with respect to citrate concentration. (C) Coupons were removed, crystal violet was solubilized and measured spectrophotometrically (A550). The average crystal violet staining of nine coupons is shown. Error bars show one SD; **P-value of <0.01.

 

Figure 2
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  		Fig. 2. Microscopic analysis of citrate exposed biofilms. Micrographs of 4 h biofilms formed on polystyrene in response to citrate concentration, shown as citrate percent (wt/vol). Images magnified 400x were observed with phase-microscopy (A, C, E) and stained with a fluorescent dye, Syto-9, that preferentially stains living bacterial cells (B, D, F). Bars = 2 µ.

 
To determine whether existing biofilms could be eliminated by the addition of sodium citrate, we allowed biofilms to form for 8 h in the absence of sodium citrate. Then we added sodium citrate (0–4%) and incubated for 16–18 h at 37°C. Remaining biofilms were then quantified. We determined that even 4% citrate did not disrupt pre-existing biofilms (data not shown).

The effect of sodium citrate on S. aureus biofilm formation was also assessed on silicone elastomer and polyurethane surfaces, polymers used in the manufacture of many catheters. When incubated with 0.2% sodium citrate, a greater than 2-fold increase in biofilm formation was observed on silicone elastomer coupons at 20 h (Figure 1B and C). However, 2 and 4% sodium citrate inhibited biofilm formation on silicone elastomer (Figure 1B and C). A similar trend was observed on polyurethane though the cells were better able to adhere to polyurethane. The adherence to polyurethane at 0% citrate determined by spectrophotometric absorbance of crystal violet staining was 0.6 ± 0.17, at 0.2% citrate adherence increased to 1.12 ± 0.09, and decreased to 0.33 ± 0.05 and 0.23 ± 0.08 at 2 and 4% citrate, respectively. In all cases, the difference between the citrate treated conditions and the absence of citrate was significantly different with P < 0.001.

We repeated the above experiments, but instead observed biofilms directly with phase-contrast and fluorescent microscopy rather than with the crystal violet microtitre dish assay (Figure 2A–F). A clear decrease in biofilm formation was seen at citrate levels above 0.25%. While at 0.125 and 0.25% sodium citrate levels, biofilm formation is greatly enhanced (Figure 2C and D). These hyper-biofilms were very thick and devoid of much of the architecture seen in untreated biofilms (see microcolony indicated by the arrow in Figure 2A). Adherent S. aureus cells were stained with Syto-9, a fluorescent dye that preferentially stains living bacterial cells, and propidium iodide (PI), which stains bacterial cells with disrupted membranes. Syto-9 and PI staining indicates that the few bacterial cells able to adhere to abiotic surfaces in the presence of 2 and 4% sodium are viable (Figure 2E, F and data not shown).

Sodium citrate inhibits S. aureus growth
The reason for decreased biofilm formation at high concentration of sodium citrate could be a consequence of growth inhibition of S. aureus by citrate. The kinetics of bacterial growth were determined by measuring culture turbidity over time in TSB + glucose + citrate at various concentrations. These are the same media conditions in which biofilm formation was assessed. We found that S. aureus growth was inhibited at 1, 2 and 4% but not at 0.2% (1% is not shown, Figure 3A). This result is consistent with the greatly reduced rates of biofilm formation at 1–4% sodium citrate being a consequence of the inhibition of population growth by citrate.


Figure 3
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  		Fig. 3. Analysis of sodium citrate's effect on S. aureus growth and biofilm formation. (A) Growth of S. aureus strain MZ100 in TSB + 0.2% glucose with 0–4% sodium citrate was observed over a period of 24 h. Optical density is shown with respect to time. All experiments were done in triplicate. Error bars show one SD. (B) Biofilm stimulation by 0.2% citrate is not reversed by the addition of divalent cations that may be chelated by citrate. The addition of 10 mM sodium, the equivalent amount of sodium in medium with 0.2% sodium citrate, in the absence of citrate does not stimulate biofilm formation, as does 0.2% citrate. The addition of 100 mM sodium, more than the amount of sodium in medium with 2% citrate, does not prevent biofilm formation, as does 2% citrate.

 
The lack of enhanced cell growth in the presence of 0.2% citrate indicates that the increase in biofilm formation is not a consequence of more bacterial cells in the culture. This suggests that citrate, rather than increasing biomass, acts either directly or indirectly to modify the way bacterial cells interact with surfaces.

Sodium citrate can be a chelator of cations such as calcium, magnesium and iron. We found that the addition of 10 or 100 mM CaCl2 or MgCl2, but not FeCl2, could rescue both culture growth and biofilm formation of S. aureus in the presence of 2% sodium citrate (data not shown). These data are consistent with sodium citrate inhibiting growth due to either depletion of cations from the growth medium or by removal of essential cations from bacterial cells. Helander and Mattila-Sandholm [11] have reported that the membranes of several Gram-negative bacteria were permeabilized by sodium citrate, and could be rescued by MgCl2, and concluded that citrate toxicity stemmed from cation chelation. Free calcium levels were assessed with fluorescent indicators at a range of citrate levels and only small variations were observed. Calcium levels were 8.6 µM at 0% citrate, 8.3 µM at 0.2%, 7.1 µM at 2.0% and 6.5 µM at 4.0% citrate. This suggests that calcium levels are not responsible for the cell death phenotype observed at 2–4% citrate.

Calcium was added to the medium supplemented with 0 and 0.2% citrate to detect whether the reduction in calcium was responsible for increased biofilm formation. Likewise, other divalent cations, manganese and magnesium, were separately added to 0.2% citrate supplemented medium to test if they could prevent stimulation of biofilm formation. Biofilm formation was still strongly stimulated by 0.2% citrate in the presence of these cations (Figure 3B). These data are consistent with the hypothesis that the citrate-dependent biofilm stimulation phenotype exhibited by S. aureus is mediated by a response to citrate levels rather than cation-depletion.

The addition of trisodium citrate clearly would increase the amount of sodium in solution, and sodium is known to effect biofilm formation. High salt levels have previously been shown to stimulate biofilm formation in S. aureus and Staphylococcus epidermidis [12,13]. Free sodium increased from 98 mM at 0% to 107 mM at 0.2% citrate, and to 156 mM at 2% citrate. To test whether the increase in biofilm formation at 0.2% citrate was due to the increase in sodium levels, we added 10 mM of sodium and found that there was no increase in biofilm formation until >100 mM was added (0 mM Na supplement yielded A550 biofilm formation of 0.85 ± 0.15, 0.78 ± 0.14 at 10 mM added sodium, 0.96 ± 0.11 at 100 mM and 1.73 ± 0.1 at 1000 mM, Figure 3B). These data show sodium at >150 mM was insufficient to prevent bacterial growth and found that cells grew well at levels of sodium added with inhibitory citrate levels (Figure 3B and data not shown). This is not surprising, as it is well known that S. aureus can withstand very high levels of NaCl.

Trisodium citrate has little effect upon the pH under our experimental conditions. The pH of medium supplemented with 0 and 0.2% citrate was 7.2, indicating that a change in pH is not the cause of biofilm stimulation at 0.2% citrate. The pH of the medium increases to 7.4–7.5 in medium supplemented with 2–4% trisodium citrate. As this pH is well tolerated by S. aureus, the hypothesis that a citrate-induced change in pH is responsible for growth inhibition is unlikely.

Sodium citrate has different effects on S. aureus and coagulase-negative staphylococci (CNS) biofilm formation
A range of S. aureus clinical isolates was tested for biofilm formation with various concentrations of sodium citrate (0, 0.2, 2 and 4%) in TSB + glucose. We assessed biofilm formation of 24 S. aureus clinical isolates from various sources, and observed that 21 were stimulated in biofilm formation at 0.2% citrate, and all were inhibited in biofilm formation at 2 and 4% citrate (Table 1). Five commonly used laboratory strains including the methicillin-resistant S. aureus (MRSA) strain COL. were also tested; all were inhibited for biofilm formation at 2 and 4%, and all but COL were stimulated by 0.2% citrate (Table 1). The average increase in biofilm formation (0.2% citrate biofilm levels above 0% biofilm levels) was 253% for all S. aureus strains tested. The maximum increase induced by 0.2% citrate was 848% (9.5-fold) above biofilms formed in the absence of citrate. The average decreases in S. aureus biofilm formation observed in the presence of 2 and 4% citrate were 79 and 63%, respectively.

Six CNS strains were also tested. None were stimulated in biofilm formation by 0.2% citrate, though all were inhibited at higher concentrations (Table 1). On average the presence of citrate inhibited CNS biofilm formation at 0.2, 2 and 4%. At these concentrations CNS biofilm formation was reduced by 40, 90 and 97%, respectively.

Sodium citrate and gentamicin together are effective at preventing high levels of biofilm formation
The combination of sodium citrate and gentamicin or other antibiotics in catheter locks has been suggested for use [14,15]. Because of this, we tested the efficacy of this combination to prevent biofilm formation. To test this, we used sodium citrate concentration of 0, 0.2, 2.0 and 4.0%, and varied gentamicin concentration from 0 to 40 mg/ml in the biofilm formation medium (TSB + 0.2% glucose). These high concentrations of gentamicin have been suggested in the literature. The ability of MZ100 to form biofilms with the addition of even the lowest concentration of gentamycin (2.5 mg/µl) was strongly inhibited (Figure 4A).


Figure 4
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  		Fig. 4. Gentamicin and citrate together strongly inhibit biofilm formation. (A) Biofilm formation of S. aureus strain MZ100 on polystyrene at 8 h when treated with a combination of sodium citrate and gentamicin. B, C and D show micrographs of S. aureus strain MZ100 biofilms formed for 4 h on polystyrene in the presence of both 2% sodium citrate and 5mg/ml gentamicin. (B) Depicts a 400x phase two micrograph, (C) shows the same field of bacterial cells stained with propidium iodide, a fluorescent dye which binds to bacterial cells with disrupted membranes (dead) and (D) depicts the same bacterial cells stained with the fluorescent dye, Syto-9, which preferentially stains living bacterial cells.

 
This effect was also assessed microscopically using phase and epifluorescent microscopy. We found a great reduction in the amount of biofilm formation in the presence of both citrate and gentamicin. However, even at the highest concentrations of both compounds (4.0% sodium citrate and 40 mg/ml gentamicin), a small number of adherent bacterial cells remained on the polystyrene surface, and these appeared to be living bacterial cells when assessed by a fluorescent Live/Dead staining procedure (Figure 4B–D and data not shown). The minimum inhibitory concentration of gentamicin for MZ100 in TSB + 0.2% glucose for planktonic bacterial cells was found to be 2.0 µg/ml which is 10 000x less gentamicin than the amount surface-associated bacterial cells appeared to be resistant to based on this observation (Figure 4 and R.M.Q. Shanks and G.A. O’Toole unpublished observations).

The effect of other anticoagulants on staphylococcal biofilm formation
A previous study has shown that sodium heparin enhances the accumulation of S. aureus on polymer surfaces [7]. LMWH is an effective anticoagulant and has been reported to be less likely to cause heparin-associated complications [16]. We tested whether LMWH incubated with S. aureus also increased adherence to polystyrene and found that it increased biofilm formation by more than 2-fold (Figure 5A).


Figure 5
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  		Fig. 5. The effect of other anticoagulants on S. aureus biofilm formation (A–D). Biofilm formation of S. aureus, strain MZ100, on polystyrene at 8 h in TSB + 0.2% glucose with several anticoagulants. Biofilms were stained with crystal violet and biofilm levels are shown on the y-axis. (A) Low molecular weight heparin (LMWH) in mg/ml. (B) EDTA in mM. (C) Alteplase or tissue plasminogen activator in mg/ml. (D) Lepirudin in mg/ml.

 
The divalent cation chelator, EDTA, could be used as an anticoagulant lock solution in catheters, and is antibacterial [17]. The effect of EDTA on S. aureus biofilm formation was determined over an EDTA concentration gradient from 0.2 to 50 mM. Growth and biofilm formation was inhibited at 0.2 mM and higher levels of EDTA (Figure 5B and data not shown).

The effect of tissue plasminogen activator (tPA) on bacterial adherence was also assessed. This recombinant human serine protease did not stimulate biofilm formation at the tested concentrations and may have slightly inhibited biofilm formation at the highest concentrations (Figure 5C). The preparation we used contained two additives; the amino acid L-arginine and the preservative polysorbate 80. These could formally be responsible for the reduction in biofilm formation seen at high concentrations of tPA.

Staphylococcus aureus bacterial cells were exposed to lepirudin, a direct thrombin inhibitor derived from leaches, and biofilm formation was determined. Lepirudin, or hirudin, is expensive, and can be used with patients that are known to have anti-heparin antibodies or those suffering from heparin-induced thrombocytopenia [20]. Lepirudin did not significantly modify S. aureus biofilm formation under these experimental conditions (Figure 5D).



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Biofilm formation is strongly influenced by environmental conditions, and biofilms form on several surfaces associated with haemodialysis [21]. Our study demonstrates that the choice of catheter lock solution is likely to have a significant effect on the ability of bacteria to adhere to surfaces. Specifically, we demonstrate that sodium citrate inhibits and heparin enhances biofilm formation on materials used in the manufacture of indwelling dialysis catheters. This observation provides a biological mechanism for the observed clinical superiority of sodium citrate over heparin as a catheter lock solution in a prospective randomized study performed in haemodialysis patients [9]. We demonstrate that LMWH stimulates the formation of S. aureus biofilms in vitro, and sodium citrate and sodium EDTA at high concentrations strongly inhibit biofilm formation. In addition, the combination of sodium citrate and gentamicin at bactericidal concentrations is very effective in reducing the formation of biofilms on abiotic surfaces. We demonstrate that two additional potential catheter lock solutions, lepirudin and tPA, have little effect on S. aureus biofilm formation.

The genesis of catheter-related infections is likely to be complex. In addition to bacterial pathogenicity, which includes, but is not limited, to biofilm formation, the development of clinically relevant infection must involve environmental factors including catheter surface characteristics, thrombosis, blood composition and immunological response. Catheter lock solutions are used for two closely related, but separate, purposes: to prevent thrombosis and to prevent infection. Heparin is an effective anticoagulant, and as such, likely contributes to the prevention of dialysis catheter infection by minimizing the local accumulation of host-factors that promote biofilm formation. At the same time, however, heparin is an independent promoter of biofilm formation. This together with the disadvantages of high cost and anti-heparin antibodies emphasizes the importance of considering alternative catheter lock solutions.

Sodium citrate is inexpensive and is very well established as an anticoagulant in transfusion medicine and in continuous veno-venous haemofiltration systems. However, sodium citrate has not gained wide acceptance for use in intermittent haemodialysis in the US primarily because of fear of accidental infusion of highly concentrated solutions of sodium citrate during the lock procedure [22].

When considering the potential for prevention of bloodstream infections, it must be noted that small numbers of viable adherent bacterial cells persist on the material surface even at the highest levels of treatment with sodium citrate and sodium citrate plus gentamicin. These could potentially serve as a source of bloodstream infection. However, it is reasonable to postulate that smaller numbers of bacteria may be more readily cleared by the immune system than larger numbers. In addition, we hypothesize that the prevention of mature biofilm formation in haemodialysis catheters may be central to the prevention of complex infections such as endocarditis, joint and soft tissue infections because these microbial communities can actively detach from surfaces. Detachment behaviour, which is genetically determined, causes rafts of living biofilm or single cells to be released from the surface with the potential to lodge in distant sites [23].

Our data demonstrate that subinhibitory concentrations of sodium citrate actually enhance biofilm formation. This finding is of potential clinical significance because the concentration of citrate, as of all catheter lock solutions, varies at the catheter tip depending on the viscosity of the injected solution [24]. Agharazii and colleagues [25] have shown that up to 75% of the catheter contents may leak in the first 30 min after performing a catheter lock. However, a clinical trial that employed 5% citrate as a catheter lock solution did not show a higher level of infection than catheters locked with 10% citrate, consistent with the increase in biofilm formation caused by low levels of citrate in vitro having little effect upon the rate of infection in clinical practice [26].

It is unclear at this point how low concentrations of sodium citrate cause this stimulation in biofilm formation, though the data presented here suggest that it is not direct chelation of calcium, magnesium or manganese ions. Sodium levels may contribute, but are not sufficient for the enhancement in biofilm formation. Currently, genetic experiments are underway to identify factors responsible for the stimulation. We have found that the SarA transcription factor is essential in this response (R.M.Q. Shanks and G.A. O’Toole, unpublished results), further suggesting that it is not a direct physical manifestation from the presence of citrate, but rather an example of bacteria sensing their environment and responding by altering their life style.

It will be of interest to test the effectiveness of sodium citrate and sodium EDTA in the clinic and on in vivo biofilm formation. There are several reports that indicate sodium citrate is as effective as a catheter lock solution in the clinic as heparin [27]. Likewise, sodium EDTA has been shown by itself or in conjunction with antibiotics to be inhibitory to the formation of biofilms in vitro and in vivo [18,19,28–32].

It has been hypothesized that the bactericidal activity of cationic chelators stems from two activities [11]. First, calcium and magnesium ions are necessary for prokaryotic cell division, and second, they are important for the integrity of the bacterial cell wall. Sodium citrate and EDTA are both capable of chelating calcium and magnesium ions leading to inhibition of bacterial growth.

In conclusion, this study shows that, in contrast to heparin and LMWH, sodium citrate at 2% concentrations or greater powerfully inhibits in vitro biofilm formation by S. aureus and CNS. At much lower concentrations, which could theoretically be achieved within or downstream from locked dialysis catheters, sodium citrate actually promotes biofilm formation. These observations, together with recent clinical studies demonstrating the efficacy of sodium citrate as a catheter lock solution in dialysis patients, raise intriguing questions about the pathogenic mechanisms of clinically significant bloodstream infections in haemodialysis patients with indwelling dialysis catheters.



   Acknowledgments
 
We are grateful to Michael Zegans, Stuart Powers and members of the O’Toole lab for helpful discussion, and to Joe Schwartzman, NARSA, Niles Donegan and Ambrose Cheung for strains. This work was sponsored by NIH grants F32 GM66658-01A1 to R.M.Q.S., and NIH AI51360-01 to G.A.O’T.

Conflicts of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 8. 9.05
Accepted in revised form: 15. 3.06


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