JAC Advance Access published online on July 18, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn291
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Original research |
Effectiveness of a 30% ethanol/4% trisodium citrate locking solution in preventing biofilm formation by organisms causing haemodialysis catheter-related infections
1 Faculty of Pharmacy, University of Manitoba, 50 Sifton Road, Winnipeg, MB, Canada R3T 2N2 2 St Boniface General Hospital, 409 Tache Avenue, Winnipeg, MB, Canada R2H 2A6 3 Manitoba Renal Program, 820 Sherbrook Street, Winnipeg, MB, Canada R3A 1R9
* Corresponding author. Tel: +1-204-474-6043; Fax: +1-204-474-7617; E-mail: lavern_vercaigne{at}umanitoba.ca
Received 5 April 2008; returned 21 May 2008; revised 11 June 2008; accepted 20 June 2008
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Abstract |
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Objectives: Antibiotic locks may be used to prevent haemodialysis catheter-related infections (CRIs). This in vitro study tested the effectiveness of a novel 30% ethanol/4% trisodium citrate lock solution in preventing biofilm formation by the most common pathogens causing haemodialysis CRIs.
Methods: Ten clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli were tested. Bacterial suspensions of each isolate were prepared in a control solution of normal saline/Mueller–Hinton broth (MHB) and in a lock solution of 30% ethanol/4% trisodium citrate and MHB. The bacterial suspensions were placed into the wells of a Calgary Biofilm Device (CBD) and incubated with fresh solution every 24 h for 72 h. The biofilm formation was assessed by removing the CBD pegs, placing them in normal saline and sonicating them for 5 min. The resulting solution was sampled and the colony counts were determined after 24 h of incubation.
Results: All controls demonstrated biofilm growth of between 6 x 106 and 7.4 x 107 cfu/mL over 72 h. In the 30% ethanol/4% trisodium citrate lock solution, no biofilm growth was observed after 72 h of incubation. These results were consistent among duplicates of all isolates.
Conclusions: The 30% ethanol/4% trisodium citrate lock solution prevented the biofilm formation of all isolates of S. aureus, S. epidermidis, P. aeruginosa and E. coli in vitro. Further studies are necessary to determine its efficacy and safety in the haemodialysis population.
Key Words: haemodialysis , vascular access , infection , catheters
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Introduction |
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Currently, haemodialysis is the most common treatment for end-stage renal disease in North America. While central venous catheters are not the preferred method of vascular access, they are still commonly used in
40% of prevalent haemodialysis patients in Canada.1 Catheter-related infection (CRI) is a major cause of morbidity and mortality.2 A recent meta-analysis has summarized the benefits of using antibiotic/anticoagulant catheter locking solutions to prevent CRIs.3 However, what remains unclear is the potential to develop resistant organisms with chronic use of antibiotic-based catheter locking solutions. As an alternative to antibiotics, we are interested in using ethanol as the antiseptic agent in haemodialysis catheters made of carbothane, thereby reducing the risk of developing antibiotic resistance with chronic exposure. Ideally, ethanol would prevent biofilm formation and prevent CRIs.
The goal of the present study was to determine whether a 30% ethanol/4% trisodium citrate solution would prevent in vitro biofilm formation by organisms most commonly associated with haemodialysis CRIs.
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Materials and methods |
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Collection of clinical isolates
Clinical isolates of methicillin-susceptible Staphylococcus aureus (MSSA) (n = 2), methicillin-resistant Staphylococcus epidermidis (MRSE) (n = 2), Pseudomonas aeruginosa (n = 2) and Escherichia coli (n = 2) were obtained from blood cultures of haemodialysis patients with catheter-related bacteraemia between 2000 and 2005. Methicillin-resistant S. aureus (MRSA) isolates (n = 2) were obtained from blood and sputum cultures of two non-haemodialysis patients because of the rare occurrence of MRSA bacteraemia in local dialysis centres.
The Calgary Biofilm Device (CBD) and the methods established by the manufacturer (MBECTM Bioproducts, Inc., Edmonton, AB, Canada) were used to test the 30% ethanol/4% trisodium citrate lock solution in preventing the biofilm formation. The CBD plate consists of 96 wells in which 96 pegs attached to the lid are immersed.4 The device is placed on a rocker producing the shear force to enhance biofilm formation during incubation.
Composition of the control and lock solutions
A 78.9% ethanol stock solution was prepared from dehydrated ethyl alcohol for injection, 7.89 g/10 mL (Sandoz Canada, Inc., Quebec, QC, Canada). A 40% trisodium citrate stock solution was prepared using 4.0 g of trisodium citrate powder from sodium citrate tribasic:dehydrate powder (Sigma-Aldrich Co., St Louis, MO, USA) in 10 mL of distilled water and adjusting to a pH of 6.4–7.5 with 1 M citric acid.
The lock solution was made with 0.95 mL of ethanol stock, 0.25 mL of trisodium citrate stock, 1.275 mL of Mueller–Hinton broth (MHB) and 25 µL of bacterial suspension with a turbidity equivalent to that of a 0.5 McFarland standard. The control solution was a mixture of 1.2 mL of normal saline, 1.275 mL of MHB and 25 µL of bacterial suspension. Fresh nutrient solution without bacteria was prepared every 24 h and added to the wells to support bacterial growth in the device.
Control and lock solutions (2.475 mL) were inoculated with 25 µL of bacterial suspension to yield initial densities of 1 x 106 cfu/mL. The CBD was filled with 250 µL of solution per well, placed on a rocker at 90 rpm and incubated at 37°C for 72 h. Every 24 h growth medium was supplemented with fresh nutrient solution.
To determine the bacterial loads on each peg after 72 h, the CBD wells were filled with 250 µL of normal saline and placed back on the rocker for 1 min to remove the residual organisms from the peg surfaces. The pegs were then broken off, placed in 0.5 mL of normal saline and sonicated for 5 min to disrupt any biofilm. Samples from the resultant solution were plated on tryptic soy agar and incubated for 72 h. Plates were inspected every 24 h for viable colonies. The lower limit of detection was 1 x 102 cfu/mL. To improve the sensitivity of detection, 100 µL of the resultant solution (following sonication) was added to 9.9 mL of MHB, incubated at 37°C for an additional 48 h and sampled for viable colonies. All isolates were tested in duplicate on separate days.
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Results |
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The results of all tests are shown in Figure 1. After incubating in the CBD for 72 h, sonicating the pegs and plating the resulting solution, all control organisms grew biofilm to 6 x 106–7.4 x 107 cfu/mL. The organisms exposed to the lock solution containing 30% ethanol/4% trisodium citrate did not produce detectable biofilm after 72 h of incubation. The addition of 100 µL of the solution remaining after sonication to 9.9 mL MHB produced no growth.
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Discussion |
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The results of this study demonstrate that the 30% ethanol/4% trisodium citrate locking solution prevents biofilm formation in vitro. This solution could potentially be locked into haemodialysis catheters between dialysis sessions to prevent CRIs. The rapid kill of organisms by ethanol will not allow bacteria to become established and will therefore prevent bacterial biofilm formation.5
Although this is the first investigation of a 30% ethanol/4% trisodium citrate locking solution to prevent biofilm formation, other investigators have studied higher concentrations of ethanol alone to treat established CRIs in non-haemodialysis patients.6–8
Dannenberg et al.6 conducted a retrospective review of catheter-related bacteraemia or sepsis in paediatric oncology patients. Patients were treated with either systemic antibiotics alone or systemic antibiotics with an ethanol lock consisting of 74% ethanol for 20–24 h. Sixty-seven percent of the patients (16/24) treated with systemic antibiotics and the ethanol lock technique experienced no new infectious events within 4 weeks (or during subsequent aplasia) compared with 47% (7/15) treated with systemic antibiotics alone.
In addition, Onland et al.7 conducted a retrospective review of 40 paediatric patients with a history of CRIs. Patients received systemic antibiotics and a 70% ethanol lock which dwelled for 12–24 h. Each catheter lumen was exposed to the locking solution for 5 days. In all 40 patients, intravascular devices were successfully retained after exposure to the ethanol lock solution. Only three patients (3/40, 7.5%) experienced recurrent bacteraemia within 30 days.
Data to support the preventive use of ethanol/anticoagulant locking solutions in the haemodialysis population are still limited. Recently, a case report documented the success of a 60% ethanol/500 IU/mL heparin lock solution between haemodialysis sessions to prevent CRIs over a period of 29 days.9
Our approach to developing the 30% ethanol/4% citrate lock solution differs from that of previous studies in several ways. The solution consists of a much lower concentration of ethanol (30%), with trisodium citrate (4%) as the anticoagulant. We are primarily interested in using the solution from the time of catheter insertion as a preventive strategy before a biofilm develops in the catheter. A 30% ethanol concentration produces a rapid kill of planktonic bacteria and should prevent biofilm formation between dialysis sessions.5
A decreased concentration of ethanol and trisodium citrate should reduce the risk of adverse events. Although no serious adverse events have been reported in ethanol lock studies to date, some mild adverse events have been reported with 70% concentrations in children, including tiredness, headaches, dizziness, nausea and light-headedness.6 Some of these adverse effects have been associated with flushing the ethanol lock into the circulation after its dwell time. Whenever possible, we suggest that the 30% ethanol/4% trisodium citrate lock be removed before each dialysis session. Clinical trials involving larger sample sizes need to be conducted before safety can be definitively established.
Although most ethanol locking studies have not included an anticoagulant in the solution, we have chosen to include a 4% trisodium citrate component. Four percent citrate has already proven to be an effective agent to maintain patency of haemodialysis catheters.10 It remains to be seen whether the combination of 30% ethanol/4% trisodium citrate will retain similar benefits in maintaining catheter patency. Increased thrombosis rates have not been reported with ethanol use in central venous catheters over the short term;6–9 thus, we do not anticipate that the addition of 30% ethanol to 4% trisodium citrate would be problematic when dwelling between haemodialysis sessions.
The current study has some limitations. The CBD is made of a different polymer compared with haemodialysis catheters, but was selected for the study because of its established methodology and ability to grow biofilm. In addition, the in vitro environment does not account for other in vivo factors that may contribute to biofilm formation (e.g. fibrin, plasma, platelets etc.) and hence limits extrapolation of the results to the in vivo setting.
We conclude that the 30% ethanol/4% trisodium citrate lock solution prevented the biofilm formation of all isolates of MRSA, MSSA, MRSE, P. aeruginosa and E. coli in vitro. Further studies are necessary to determine its efficacy and safety in the haemodialysis population.
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The Manitoba Renal Program, Renal Research and Development Committee supported this initiative.
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None to declare.
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References |
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1 . Canadian Institute for Health Information. 2007 CORR Report—Treatment of End-Stage Organ Failure in Canada, 1996–2005 (2008) http://www.cihi.ca/cihiweb/dispPage.jsp?cw_page=AR_5_E (3 June 2008, date last accessed). Ottawa, Canada: Canadian Institute for Health Information.
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Astor BC, Eustace JA, Powe NR, et al. Type of vascular access and survival among incident hemodialysis patients: the Choices for Healthy Outcomes in Caring for ESRD (CHOICE) Study. J Am Soc Nephrol (2005) 16:1449–55.
3 . Jaffer Y, Selby NM, Taal MW, et al. A meta-analysis of hemodialysis catheter locking solutions in the prevention of catheter-related infection. Am J Kidney Dis (2008) 51:233–41.[CrossRef][Web of Science][Medline]
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Ceri H, Olson ME, Stremick C, et al. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol (1999) 37:1771–6.
5 . Takla TA, Zelenitsky SA, Vercaigne LM. Effect of ethanol/trisodium citrate lock on microorganisms causing hemodialysis catheter-related infections. J Vasc Access (2007) 8:262–7.[Web of Science][Medline]
6 . Dannenberg C, Bierbach U, Rothe A, et al. Ethanol-lock technique in the treatment of bloodstream infections in pediatric oncology patients with broviac catheter. J Pediatr Hematol Oncol (2003) 25:616–21.[CrossRef][Web of Science][Medline]
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Onland W, Shin CE, Fustar S, et al. Ethanol-lock technique for persistent bacteremia of long-term intravascular devices in pediatric patients. Arch Pediatr Adolesc Med (2006) 160:1049–53.
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Opilla MT, Kirby DF, Edmond MB. Use of ethanol lock therapy to reduce the incidence of catheter-related bloodstream infections in home parenteral nutrition patients. J Parenter Enteral Nutr (2007) 31:302–5.
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Ackoundou-N'guessan C, Heng AE, Guenu S, et al. Ethanol lock solution as an adjunct treatment for preventing recurrent catheter-related sepsis—first case report in dialysis setting. Nephrol Dial Transplant (2006) 21:3339–40.
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Lok CE, Appleton D, Bhola C, et al. Trisodium citrate 4%—an alternative to heparin capping of haemodialysis catheters. Nephrol Dial Transplant (2007) 22:477–83.
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