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In Vitro Assessment of the Antimicrobial Efficacy of Optimized Nitroglycerin-Citrate-Ethanol as a Nonantibiotic, Antimicrobial Catheter Lock Solution for Prevention of Central Line-Associated Bloodstream Infections Ruth A. Reitzel,a Joel Rosenblatt,a Cheryl Hirsh-Ginsberg,b Kimberly Murray,c Anne-Marie Chaftari,a Ray Hachem,a Issam Raada Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAa; Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAb; School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAc
The rapid, broad-spectrum, biofilm-eradicating activity of the combination of 0.01% nitroglycerin, 7% citrate, and 20% ethanol and its potential as a nonantibiotic, antimicrobial catheter lock solution (ACLS) were previously reported. Here, a nitroglycerincitrate-ethanol (NiCE) ACLS optimized for clinical assessment was developed by reducing the nitroglycerin and citrate concentrations and increasing the ethanol concentration. Biofilm-eradicating activity was sustained when the ethanol concentration was increased from 20 to 22% which fully compensated for reducing the citrate concentration from 7% to 4% as well as the nitroglycerin concentration from 0.01% to 0.0015% or 0.003%. The optimized formulations demonstrated complete and rapid (2 h) eradication of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant enterococci (VRE), multidrug-resistant (MDR) Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, MDR Enterobacter cloacae, MDR Acinetobacter baumannii, MDR Escherichia coli, MDR Stenotrophomonas maltophilia, Candida albicans, and Candida glabrata biofilms. The optimized NiCE lock solutions demonstrated anticoagulant activities comparable to those of heparin lock solutions. NiCE lock solution was significantly more effective than taurolidine-citrate-heparin lock solution in eradicating biofilms of Staphylococcus aureus and Candida glabrata. The optimized, nonantibiotic, heparin-free NiCE lock solution demonstrates rapid broad-spectrum biofilm eradication as well as effective anticoagulant activity, making NiCE a high-quality ACLS candidate for clinical assessment.
C
entral line-associated bloodstream infections (CLABSIs) pose a significant public health problem in the United States. More than 7 million patients require central venous access each year, resulting in an estimated 250,000 CLABSIs, approximately 30,000 attributable deaths (1), and substantial treatment costs of at least $45,000 per infection (2). The majority of CLABSIs occur in chronically catheterized patients, such as cancer, hemodialysis, or critical care patients. Long-term catheters, such as central venous catheters (CVCs), peripherally inserted central catheters (PICCs), ports, and dialysis catheters, are susceptible to internal colonization of the catheter lumens due to repeated catheter manipulation and infusions. Biofilm formation on the catheter surfaces can serve as the primary source of CLABSI and can be highly resistant to eradication by systemic antibiotic therapy. Several bundled measures have been proposed for both treatment and prevention of CLABSIs, most of which center around reducing and preventing colonization at the skin insertion site or at connections (3). While bundles have been shown to decrease the risk of CLABSI in short-term CVCs (⬍30 days of dwell time), approximately 90% of CLABSIs in longer-term catheters are luminally sourced (4, 5). While antimicrobial coatings exist that do cover luminal surfaces, the duration of protection is limited to only a few weeks (6). Because disinfecting antimicrobial catheter lock solutions (ACLSs) can be repetitively instilled, they have emerged as another approach to prevent luminally sourced CLABSIs. Antimicrobial lock therapy solutions for preventing CLABSI have been reported to include prophylactic applications of antibiotics, antifungals, or antiseptics such as ethanol (EtOH) (7, 8). The
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development of microbial resistance is of great concern with prophylactic antibiotic ACLSs (9). Alternatively, high-concentration antiseptic ethanol ACLSs have been studied. An ACLS consisting of 70% ethanol was not effective in decreasing intraluminal CLABSI and was associated with adverse events such as dizziness (10). Ethanol ACLSs with concentrations of greater than 30% have also been associated with clotting, transient dizziness, and compromising catheter integrity in polyurethane catheters (11). Use of ethanol locks with ethanol concentrations greater than 28% has been further advised against due to the significant potential for protein precipitation at the blood-lock solution interface (12). The in vitro biofilm eradication model highly correlated with clinical microbiological eradication from catheter cultures obtained from CLABSI patients that had their catheters locked for 2 h daily with a minocycline-EDTA-ethanol lock solution (13). We reported the efficacy of 0.01% glyceryl trinitrate (GTN; nitroglyc-
Received 4 February 2016 Returned for modification 23 February 2016 Accepted 4 June 2016 Accepted manuscript posted online 13 June 2016 Citation Reitzel RA, Rosenblatt J, Hirsh-Ginsberg C, Murray K, Chaftari A-M, Hachem R, Raad I. 2016. In vitro assessment of the antimicrobial efficacy of optimized nitroglycerin-citrate-ethanol as a nonantibiotic, antimicrobial catheter lock solution for prevention of central line-associated bloodstream infections. Antimicrob Agents Chemother 60:5175–5181. doi:10.1128/AAC.00254-16. Address correspondence to Joel Rosenblatt, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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erin)–7% citrate–20% ethanol as a potential ACLS. Complete eradication of biofilms formed by selected key multidrug-resistant (MDR) organisms within 2 h of exposure were reported, indicating that the combination of nitroglycerin-citrate-ethanol (NiCE) could be a practical candidate for a preventative ACLS (14). In order to optimize a formulation for clinical testing, we adjusted the concentrations of the formulation. In 2000, the U.S. Food and Drug Administration (FDA) issued a warning that concentrated trisodium citrate should not be used as an ACLS due to a reported death from cardiac arrest after a mistaken bolus injection of 46.7% citrate. The FDA further advised that 4% citrate should be used in these settings. Additionally, 4% sodium citrate has been approved for use in blood collection bags for apheresis or blood transfusion where blood/citrate solution is reintroduced into circulation. We reduced the concentration of citrate from 7% to 4% due to these regulatory precedents (15, 16). If the catheter lumen is flushed, the use of nitroglycerin in a lock solution creates the potential for hypotension as a side effect, so minimizing it would be desirable. Although a 0.01% GTN concentration was below bolus doses of nitroglycerin that had been previously used safely (17–20), to further increase safety margins for potential hypotension, we reduced the concentrations of GTN to 0.0015% or 0.003%. The aim of this study was to assess how much the concentration of ethanol would need to be increased to maintain antimicrobial potency as a result of reducing the GTN and citrate concentrations. MATERIALS AND METHODS Bacterial strains. A broad spectrum of highly virulent Gram-positive, Gram-negative, and yeast pathogens from our hospital was used for testing. These strains included methicillin-resistant Staphylococcus aureus (MRSA; strain MDA 120), vancomycin-intermediate Staphylococcus aureus (VISA; MDA 104), methicillin-resistant Staphylococcus epidermidis (MRSE; MDA 146), vancomycin-resistant enterococci (VRE; MDA 119), multidrug-resistant (MDR) Pseudomonas aeruginosa (MDA 118), MDR Klebsiella pneumoniae (MDA 144), MDR Enterobacter cloacae (MDA 134), MDR Acinetobacter baumannii (MDA 133), MDR Escherichia coli (MDA 141), MDR Stenotrophomonas maltophilia (MDA 140), Candida albicans (MDA 117), and Candida glabrata (MDA 115). All pathogens tested were clinical isolates selected from the MD Anderson Infectious Diseases laboratory stock of stored organisms from cancer patient cultures. Fresh organisms were grown on Trypticase soy agar–5% sheep blood (for bacteria) or Sabouraud dextrose agar (for yeast) from glycerol stock overnight at 37°C. For testing, pure culture was inoculated into Muller-Hinton broth (MHB) and diluted to a 0.5 McFarland standard. Additional dilutions were made as necessary for testing. Assessment of antimicrobial efficacy in a biofilm eradication model. In vitro antimicrobial assessments of the optimized NiCE lock solutions were conducted using an established biofilm eradication model, modified from Kuhn et al. (21). Briefly, 1-cm-diameter silicone discs were placed into a 24-well tissue culture plate and incubated with human plasma at 37°C for 24 h. The plasma was then removed, replaced with 1 ml of 5.5 ⫻ 105 CFU of various bacteria and yeast inocula, and incubated at 37°C for an additional 24 h. Inoculum was removed, and the discs were washed with shaking at 100 rpm for 30 min in 0.9% sterile saline in order to remove any planktonic organisms. After the washing step, discs were exposed to 1 ml of various lock solutions and incubated at 37°C for 2 h. The 2-h exposure time was selected because in our hospital 2 h was the longest duration we found that we could reliably remove vascular access lines from use in critically ill cancer patients. Subsequently, discs were removed and placed in 5 ml of 0.9% sterile saline and sonicated (60 Hz and 150 W) for 15 min to disrupt any remaining biofilm. The resulting solutions were quantitatively cultured by plating 100 l onto Trypticase soy agar–5%
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sheep blood for bacterial pathogens or onto Sabouraud dextrose agar for yeast pathogens. All biofilm eradication experiments were performed with six replicates. Optimization of nitroglycerin-citrate-ethanol lock solution. The synergy of the triple combination of agents in the NiCE lock solution was further investigated to determine the optimal concentrations of nitroglycerin and ethanol in the triple combination (nitroglycerin [Baxter, Deerfield IL], citrate [Sigma-Aldrich, St. Louis, MO], ethanol [Sigma-Aldrich, St. Louis, MO]) against MRSA and VRE biofilms. These organisms were selected for this assessment because of the reported resistance of their biofilms to ethanol (22). Citrate was fixed at 4% due to regulatory precedent (15, 16), concentrations of nitroglycerin were reduced to 0.0015% and 0.003%, and ethanol was tested above 20% in 2% increments to determine the lowest concentration of ethanol needed to compensate for the reduction in nitroglycerin. Our previous study (14) showed that at a 20% ethanol concentration, a nitroglycerin concentration of 0.005% was unable to fully eradicate MRSA biofilm in 2 h (14). Hence, the first incremental step increase in ethanol concentration tested here was 22% ethanol. After optimization, NiCE combinations (0.0015% or 0.003% nitroglycerin plus 4% citrate and 22% ethanol) were compared to the previously published 0.01% nitroglycerin–7% citrate–20% ethanol lock solution in the biofilm eradication model against a broad spectrum of CLABSI pathogens. Discs exposed to Mueller-Hinton broth (MHB) were used as positive controls. Additionally, both concentrations of NiCE lock were tested for efficacy against planktonic bacteria in a broth dilution model. Briefly, fresh MRSA, P. aeruginosa, and C. albicans inocula at a 0.5 McFarland standard were diluted to 1.0 ⫻ 106 CFU/ml in MHB. A total of 500 l of each inoculum was exposed for 2 h to 500 l of NiCE lock with concentrations adjusted so that the 1-ml total volume resulted in 0.0015% or 0.003% nitroglycerin– 4% citrate–22% ethanol. After exposure, 100 l was serially diluted and quantitatively cultured for growth on Trypticase soy agar–5% sheep blood (bacteria) or Sabouraud dextrose agar (yeast). Plates were incubated for 24 h at 37°C and enumerated for growth. All solutions were tested in triplicate; MHB was tested as a control solution. Antimicrobial efficacy of optimized NiCE solution compared to taurolidine-citrate-heparin. The antimicrobial efficacy of the optimized NiCE lock solution was compared in the in vitro model to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin to further assess its suitability for clinical testing. Taurolidine-citrate-heparin lock was chosen as a comparator due to an extensive body of clinical trial literature (23, 24) that would enable conclusions to be inferred about comparative clinical efficacy of NiCE lock solution. Taurolidine-citrate-heparin lock solution was prepared from the individual components (taurolidine [Santa Cruz Biotechnology, Dallas, TX], citrate [Sigma-Aldrich, St. Louis, MO], heparin [Fresenius Kabi USA, Lake Zurich, IL]), resulting in the 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin triple combination lock solution. In these experiments optimized NiCE and taurolidine-citrate-heparin lock solutions as well as MHB controls were tested against a broad spectrum of CLABSI pathogens, previously listed above. Antimicrobial efficacy of optimized NiCE lock solution in a luminal biofilm model. To examine the antimicrobial efficacy of optimized NiCE lock as an ACLS, we examined the efficacy of NiCE lock in a modified luminal biofilm model (25–27). Inocula containing a 0.5 McFarland standard of MRSA, P. aeruginosa, VRE, and C. albicans were diluted to 1.0 ⫻ 106 CFU/ml in Muller-Hinton broth (BD, Sparks, MD) and locked with the appropriate priming volume (approximately 0.5 ml) in each lumen of a 5-French double-lumen peripherally inserted central catheter (PICC) (PowerPICC; Bard Access Systems, Salt Lake City, UT). In addition to locking the catheter with extension line clamps, the distal ends of all catheters were clamped to seal. Catheters were incubated for 24 h at 37°C. The inoculum was subsequently drained from each lumen, and the lumens were flushed with 0.9% saline to remove any unattached organisms. Either 0.0015% or 0.003% NiCE lock solution was instilled into each of the
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Optimized Nitroglycerin-Citrate-Ethanol and CLABSI
FIG 1 Synergy of optimized NiCE lock with 0.0015% or 0.003% GTN– 4% citrate–22% ethanol. For both MRSA (MDA 120) and VRE (MDA 119), the triple combination of optimized NiCE lock shows complete eradication of bacterial biofilm within 2 h of exposure. Individual components showed limited eradication within 2 h of exposures. The binary 22% ethanol– 4% citrate combination showed limited eradication of the MRSA biofilm within a 2-h exposure. This indicates that nitroglycerin, citrate, and ethanol interact synergistically in enhancing MRSA biofilm eradication. Results with nitroglycerin-citrate binary combinations were unchanged from those of MHB controls (data not shown), and results with nitroglycerin-ethanol binary combinations were unchanged from those with 22% ethanol (data not shown).
catheter lumens, which were clamped to lock at the proximal and distal ends of the catheter, and incubated at 37°C for 2 h. After exposure, all lock solutions were collected from catheters and cultured to determine the viability of organisms within the flushed lock solution. Catheters were then cut into 30-cm segments and sonicated for 15 min in 5 ml of 0.9% sterile saline. After sonication, the resulting solution was vortexed briefly and serially diluted, and 100 l was quantitatively cultured onto Trypticase soy agar–5% sheep blood (for bacteria) and Sabouraud dextrose agar (for yeast). All plates were incubated for 24 h at 37°C and enumerated for growth. Two catheters were tested for each organism for each test solution; each catheter was then cut into three, 3-cm-long segments for testing. Muller-Hinton broth was locked into catheters as a control solution. Assessment of anticoagulation. Prothrombin time (PT) was tested in the Clinical Chemistry Laboratory at MD Anderson according to standard methods (28). NiCE lock solutions as well as saline and 200 units (U)/ml heparin were tested for anticoagulant activity by PT. Heparin at a concentration of 200 U/ml was selected as a control because of its current use in our hospital as standard practice for preventing intraluminal thrombus formation in vascular catheters. PTs for 10 replicates per lock solution were measured. Statistical analyses. Student’s t test was used for all comparisons. Our hypothesis was that the average number of CFU recovered from the NiCE lock would be less than that of all other comparators. Therefore, all tests were one sided, and a P value of less than 0.05 (P ⱕ 0.05) was utilized to determine statistical significance. Statistical analyses were performed using Microsoft Office Excel 2013 (Microsoft, Redmond, WA).
RESULTS
Optimization and assessment of antimicrobial efficacy of optimized NiCE lock solution. Results for individual components of the NiCE lock solution (22% ethanol, 4% citrate, and 0.0015% and 0.003% nitroglycerin) and the binary combination of 22% ethanol– 4% citrate for antimicrobial efficacy against MRSA and VRE biofilms are presented in Fig. 1. Triple combinations of
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0.0015% nitroglycerin– 4% citrate–22% ethanol and 0.003% nitroglycerin– 4% citrate–22% ethanol produced complete biofilm eradication of MRSA and VRE following a 2-h exposure. In comparison, the single components were unable to fully eradicate these biofilms (Fig. 1). The 4% citrate–22% ethanol binary combination was able to fully eradicate the VRE biofilm but was unable to fully eradicate the MRSA biofilm. Both the binary combinations of nitroglycerin-citrate and nitroglycerin-ethanol had no effect on VRE and MRSA biofilms (data not shown). Results presented in Table 1 indicate that an ACLS with 22% ethanol and reduced citrate (from 7% to 4%) and nitroglycerin (from 0.01% to 0.0015% or 0.003%) concentrations did not diminish the NiCE ACLS effectiveness in rapidly eradicating (within 2 h) biofilms for a broad spectrum of multidrug-resistant bacterial and yeast pathogens. The slight increase in ethanol concentration was able to compensate for the substantial decreases in nitroglycerin and citrate concentrations. Further incremental increases in ethanol concentration were not required, and so were not tested. When tested against planktonic organisms (MRSA, P. aeruginosa, and C. albicans), NiCE lock demonstrated complete killing, with no viable organisms recovered for each strain tested following a 2-h exposure. This result was identical to the results seen with biofilm (Table 1). Muller-Hinton broth controls showed no change in the amount of organisms initially inoculated into the model, approximately 1.0 ⫻ 106 CFU. Comparison of antimicrobial activities of NiCE and taurolidine-citrate-heparin lock solutions. Figure 2 presents results for taurolidine-citrate-heparin and NiCE lock solutions for VISA and C. glabrata biofilms. The other organisms were also comparatively tested, and both lock solutions produced complete eradication of those biofilms within 2 h. The ability of NiCE lock solution to rapidly eradicate biofilm was significantly superior
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TABLE 1 Antimicrobial activity of NiCE lock solutions with 0.0015%, 0.003%, and 0.01% nitroglycerin Antimicrobial activity (CFU/disc) of:a Organism
MHB control (CFU/disc)
0.0015% GTN–4% citrate–22% EtOH
0.003% GTN–4% citrate–22% EtOH
0.01% GTN–7% citrate–20% EtOH
MRSA VISA MRSE VRE P. aeruginosa K. pneumoniae E. cloacae A. baumannii E. coli S. maltophilia C. albicans C. glabrata
5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
a
All organisms tested were allowed to form biofilms and then exposed to NiCE lock solutions for 2 h. They were subsequently quantitatively cultured to determine efficacy of biofilm eradication. Lower concentrations of nitroglycerin and 4% citrate NiCE lock solutions with 22% ethanol showed no difference compared to previously published results with 0.01% GTN–7% citrate–20% ethanol NiCE lock solution.
at either nitroglycerin concentration to that of taurolidinecitrate-heparin lock solution against VISA (P ⫽ 0.004) and C. glabrata (P ⫽ 0.04) biofilms and was similarly effective against the other pathogens. Antimicrobial efficacy of optimized NiCE lock solution in a luminal biofilm model. Efficacy of NiCE lock after a 2-h exposure to a microbial biofilm in the lumen of a 5-French double-lumen PowerPICC catheter is presented in Fig. 3. For all organisms tested (MRSA, P. aeruginosa, VRE, and C. albicans), both 0.0015% and 0.003% NiCE lock solutions showed significant efficacies compared to the efficacy of the control (P ⬍ 0.05 for the organism tested) in fully eradicating a luminal biofilm within 2 h of exposure to lock (Fig. 3). Additionally, there were no viable organisms cultured from flushed NiCE lock solution after a 2-h lock, in con-
trast to ⬎1 ⫻ 104 CFU cultured from the MHB control lock. This indicates that NiCE lock kills organisms in luminal biofilm rather than simply dispersing live organisms from the biofilm into solution. Assessment of anticoagulation. PT testing showed that the NiCE lock was comparable to 200 IU/ml heparin in inhibiting blood clotting. All replicates for 200 IU/ml heparin, 0.003% NiCE lock, and 0.0015% NiCE lock inhibited clotting for ⬎70 s (the upper limit of assay). Clotting times for the saline controls ranged from 23 s to 24.2 s (Table 2). DISCUSSION
Reducing the nitroglycerin concentration in NiCE lock solutions by 70 to 85% provides an additional margin of safety against tran-
FIG 2 Comparison of the antimicrobial efficacies of optimized NiCE lock to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin. Both 0.0015% and 0.003% NiCE lock solutions show significantly greater eradication of biofilm (within 2 h) than 1.35% taurolidine–3.5% citrate-1,000 U/ml heparin lock solution for both VISA (MDA 104) (P ⫽ 0.004), and C. glabrata (MDA 115) (P ⫽ 0.04). NiCE lock and taurolidine-citrate-heparin lock showed equivalent antimicrobial efficacies for all other organisms tested.
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Optimized Nitroglycerin-Citrate-Ethanol and CLABSI
FIG 3 Antimicrobial efficacy of NiCE lock in a luminal biofilm model. Both 0.0015% and 0.003% NiCE lock solutions fully eradicated MRSA, P. aeruginosa, VRE, and Candida luminal biofilms within 2 h of locking. Control catheters were locked with Muller-Hinton broth.
sient hypotension when lumens are flushed. To compensate for the reduced nitroglycerin concentration, only a slight increase of 2% in ethanol concentration (from 20 to 22%) was needed to sustain antimicrobial efficacy against the most significant CLABSI pathogens. The 22% ethanol concentration is still lower than ethanol concentrations previously tested clinically and below concentrations (⬎30%) that have been implicated in transient adverse events and catheter dysfunction (11). The concentration of citrate was reduced to 4% (from 7%) due to regulatory precedent (15, 16). At the 4% citrate concentration, antimicrobial efficacy was retained (Table 1), and anticoagulant properties were comparable to those of 200 IU/ml heparin (Table 2). Taurolidine lock solutions have been evaluated in clinical trials for prevention of CLABSI, with mixed clinical results. Some trials reported significant decreases in CLABSI versus results with hep-
TABLE 2 Assessment of anticoagulant properties for optimized NiCE lock by prothrombin time measurement PT of lock solution (s)a Replicate Saline 200 U/ml 0.0015% GTN–4% 0.003% GTN–4% no. concn (%) heparin citrate–22% EtOH citrate–22% EtOH 1 2 3 4 5 6 7 8 9 10
23.5 23.0 23.8 23.2 24.2 23 23.6 23.1 23.6 23.4
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
a Both 0.0015% and 0.003% optimized NiCE lock solutions gave ⬎70 s of clotting time (the assay limit), which was the same result as that with 200 U/ml heparin. The heparin lock concentration of 200 U/ml is normally used in our hospital for maintaining catheter patency. This result supports the idea that the 4% citrate contained in the NiCE lock provides sufficient anticoagulant activity to prevent clotting in the catheter lumens during locking intervals. PT, prothrombin time.
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arin locks (29), and others did not (23). In a multicenter, doubleblind, randomized, controlled clinical trial in hemodialysis patients, Solomon et al. (23) reported that 1.35% taurolidine–3.5% citrate (TC) lock solution did not significantly decrease bacteremia compared to the level in catheters locked with heparin (23). Catheters locked with taurolidine-citrate also had problems with clotting and showed a significant increase in the need for thrombolytic therapy (P ⫽ 0.008). In a subsequent trial, Solomon et al. (30) added heparin to taurolidine-citrate and evaluated 1.35% taurolidine–3.5% citrate–500 U/ml heparin in 106 hemodialysis patients. This addition of heparin significantly increased time to first use of thrombolytics (hazard ratio of taurolidine-citrate-heparin to taurolidine-citrate, 0.2; 95% confidence interval [CI], 0.06 to 0.5) with a nonsignificant change in time to bacteremia (hazard ratio of taurolidine-citrate-heparin to heparin, 0.4; 95% CI, 0.2 to 1.0) (30). The addition of heparin to a lock solution has two significant disadvantages. One is the significant risk of heparin allergy and heparin-associated thrombocytopenia, which reportedly can occur in 10 to 30% of patients receiving heparin (31). A second is that heparin has been shown to promote the formation of staphylococcal (32) and Candida (33) biofilms. Staphylococcal biofilm was promoted by heparin in a dose-dependent manner (34). This is particularly relevant because of the high concentrations of heparin in taurolidine-citrate-heparin required to overcome the prothrombotic tendencies of taurolidine-citrate. A clinical trial conducted in a pediatric cancer population evaluated taurolidine-citrate-heparin versus heparin (35). This study reported a significant reduction in catheter-related bloodstream infections (CRBSIs) with the use of taurolidine-citrate-heparin (0.1 CRBSIs per 1,000 CVC days with taurolidine-citrate-heparin versus 0.9 CRBSIs per 1,000 CVC days with heparin; P ⫽ 0.03); however, while taurolidine-citrate-heparin reduced the rate of CRBSI in this study, intraluminal microbial colonization was detected in most of the taurolidine-citrate-heparinlocked catheters (35). In fact, over 88% of the taurolidine-citrateheparin-locked catheters were colonized with biofilms (23 of 26
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catheters) covering a mean of 80% of the catheter surface. There was no significant difference in catheter colonization rates between the heparin and taurolidine-citrate-heparin groups. In our in vitro study, we compared optimized NiCE lock solutions (0.0015 to 0.003% GTN– 4% citrate–22% EtOH) to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin for their abilities to eradicate biofilm formed on silicone surfaces. For the majority of the common CLABSI pathogens tested, the NiCE and taurolidinecitrate-heparin lock solutions performed comparably. However, the optimized NiCE lock was superior to taurolidine-citrate-heparin against vancomycin-intermediate Staphylococcus aureus and Candida glabrata biofilms, such that taurolidine-citrate-heparin failed to eradicate these organisms while the NiCE lock achieved complete eradication (Fig. 2). Since catheter colonization is reportedly a prelude to infection (36), NiCE lock therefore has the potential to be similar to or more clinically effective than taurolidine-citrate-heparin in preventing CLABSI. The optimized NiCE lock solutions had anticoagulant properties comparable to those of heparin without the disadvantages of containing heparin. Future planned studies include a human clinical trial assessing the safety and effectiveness of the optimized NiCE lock solutions in catheterized cancer patients. ACKNOWLEDGMENTS We thank Hewan Gebreselassie and Bette Muthomi at The MD Anderson Cancer Center, School of Health Professions, for their assistance with anticoagulation testing.
REFERENCES 1. Klevens RM, Edwards JR, Richards CL, Jr, Horan TC, Gaynes RP, Pollock DA, Cardo DM. 2007. Estimating health care-associated infections and deaths in U.S. hospitals, 2002. Public Health Rep 122:160 –166. 2. Warren DK, Quadir WW, Hollenbeak CS, Elward AM, Cox MJ, Fraser VJ. 2006. Attributable cost of catheter-associated bloodstream infections among intensive care patients in a nonteaching hospital. Crit Care Med 34:2084 – 2089. http://dx.doi.org/10.1097/01.CCM.0000227648.15804.2D. 3. Frasca D, Dahyot-Fizelier C, Mimoz O. 2010. Prevention of central venous catheter-related infection in the intensive care unit. Crit Care 14: 212. http://dx.doi.org/10.1186/cc8853. 4. Raad I, Chaftari AM. 2014. Advances in prevention and management of central line-associated bloodstream infections in patients with cancer. Clin Infect Dis 59(Suppl 5):S340 –S343. http://dx.doi.org/10.1093/cid /ciu670. 5. Mermel LA, Allon M, Bouza E, Craven DE, Flynn P, O’Grady NP, Raad II, Rijnders BJ, Sherertz RJ, Warren DK. 2009. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America. Clin Infect Dis 49:1– 45. http://dx.doi.org/10.1086/599376. 6. Raad I, Reitzel R, Jiang Y, Chemaly RF, Dvorak T, Hachem R. 2008. Anti-adherence activity and antimicrobial durability of anti-infectivecoated catheters against multidrug-resistant bacteria. J Antimicrob Chemother 62:746 –750. http://dx.doi.org/10.1093/jac/dkn281. 7. Maiefski M, Rupp ME, Hermsen ED. 2009. Ethanol lock technique: review of the literature. Infect Control Hosp Epidemiol 30:1096 –1108. http://dx.doi.org/10.1086/606162. 8. Segarra-Newnham M, Martin-Cooper EM. 2005. Antibiotic lock technique: a review of the literature. Ann Pharmacother 39:311–318. http://dx .doi.org/10.1345/aph.1E316. 9. Landry DL, Braden GL, Gobeille SL, Haessler SD, Vaidya CK, Sweet SJ. 2010. Emergence of gentamicin-resistant bacteremia in hemodialysis patients receiving gentamicin lock catheter prophylaxis. Clin J Am Soc Nephrol 5:1799 –1804. http://dx.doi.org/10.2215/CJN.01270210. 10. Slobbe L, Doorduijn JK, Lugtenburg PJ, El Barzouhi A, Boersma E, van Leeuwen WB, Rijnders BJ. 2010. Prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: a randomized, placebo-controlled trial. PLoS One 5:e10840. http://dx.doi.org /10.1371/journal.pone.0010840.
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11. Mermel LA, Alang N. 2014. Adverse effects associated with ethanol catheter lock solutions: a systematic review. J Antimicrob Chemother 69: 2611–2619. http://dx.doi.org/10.1093/jac/dku182. 12. Schilcher G, Schlagenhauf A, Schneditz D, Scharnagl H, Ribitsch W, Krause R, Rosenkranz AR, Stojakovic T, Horina JH. 2013. Ethanol causes protein precipitation—new safety issues for catheter locking techniques. PLoS One 8:e84869. http://dx.doi.org/10.1371/journal.pone .0084869. 13. Raad I, Chaftari AM, Zakhour R, Jordan J, Al Hamal Z, Jiang Y, Yousif A, Garoge K, Mulanovich V, Viola G, Kanj S, Pravinkumar SE, Rosenblatt J, Hachem R. 2015. Successful salvage of central venous catheters in the setting of catheter related or central line-associated bloodstream infections using catheter lock consisting of minocycline, EDTA, and 25% ethanol, abstr 285. Abstr IDWeek 2015, San Diego, CA, 7 to 11 October 2015. Infectious Diseases Society of America, Arlington, VA. 14. Rosenblatt J, Reitzel R, Dvorak T, Jiang Y, Hachem RY, Raad II. 2013. Glyceryl trinitrate complements citrate and ethanol in a novel antimicrobial catheter lock solution to eradicate biofilm organisms. Antimicrob Agents Chemother 57:3555–3560. http://dx.doi.org/10 .1128/AAC.00229-13. 15. Food and Drug Administration. 2000. Anticoagulant sodium citrate 4% w/v solution, U.S.P.—summary basis of approval. Food and Drug Administration, Silver Spring, MD. http://www.fda.gov/BiologicsBloodVaccines /BloodBloodProducts/ApprovedProducts/NewDrugApplicationsNDAs /ucm082845.htm. 16. U.S. Food and Drug Administration. 2000. FDA issues warning on tricitrasol dialysis catheter anticoagulant. FDA talk paper T00-16. U.S. Department of Health and Human Services, Rockville, MD. 17. Mercier FJ, Dounas M, Bouaziz H, Lhuissier C, Benhamou D. 1997. Intravenous nitroglycerin to relieve intrapartum fetal distress related to uterine hyperactivity: a prospective observational study. Anesth Analg 84: 1117–1120. http://dx.doi.org/10.1097/00000539-199705000-00030. 18. Chedraui PA, Insuasti DF. 2003. Intravenous nitroglycerin in the management of retained placenta. Gynecol Obstet Invest 56:61– 64. http://dx .doi.org/10.1159/000072734. 19. Kurz DJ, Naegeli B, Bertel O. 2000. A double-blind, randomized study of the effect of immediate intravenous nitroglycerin on the incidence of postprocedural chest pain and minor myocardial necrosis after elective coronary stenting. Am Heart J 139:35– 43. http://dx.doi.org/10.1016/S0002 -8703(00)90306-5. 20. Nashed AH, Allegra JR, Larsen S, Horowitz M. 1994. Bolus i.v. nitroglycerin treatment of ischemic chest pain in the ED. Am J Emerg Med 12:288 –291. http://dx.doi.org/10.1016/0735-6757(94)90140-6. 21. Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. 2002. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 46:1773–1780. http://dx.doi.org/10.1128/AAC.46.6.1773-1780 .2002. 22. Redelman CV, Maduakolam C, Anderson GG. 2012. Alcohol treatment enhances Staphylococcus aureus biofilm development. FEMS Immunol Med Microbiol 66:411– 418. http://dx.doi.org/10.1111/1574-695X.12005. 23. Solomon LR, Cheesbrough JS, Ebah L, Al-Sayed T, Heap M, Millband N, Waterhouse D, Mitra S, Curry A, Saxena R, Bhat R, Schulz M, Diggle P. 2010. A randomized double-blind controlled trial of taurolidine-citrate catheter locks for the prevention of bacteremia in patients treated with hemodialysis. Am J Kidney Dis 55:1060 –1068. http://dx.doi .org/10.1053/j.ajkd.2009.11.025. 24. Simon A, Ammann RA, Wiszniewsky G, Bode U, Fleischhack G, Besuden MM. 2008. Taurolidine-citrate lock solution (TauroLock) significantly reduces CVAD-associated grampositive infections in pediatric cancer patients. BMC Infect Dis 8:102. http://dx.doi.org/10.1186/1471-2334 -8-102. 25. Luther MK, Mermel LA, LaPlante KL. 2016. Comparison of telavancin and vancomycin lock solutions in eradication of biofilm-producing staphylococci and enterococci from central venous catheters. Am J Health Syst Pharm 73:315–321. http://dx.doi.org/10.2146/ajhp150029. 26. LaPlante KL, Mermel LA. 2007. In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model. Nephrol Dial Transplant 22:2239 –2246. http://dx.doi.org /10.1093/ndt/gfm141. 27. Shah CB, Mittelman MW, Costerton JW, Parenteau S, Pelak M, Arsenault R, Mermel LA. 2002. Antimicrobial activity of a novel catheter
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28.
29.
30.
31. 32.
lock solution. Antimicrob Agents Chemother 46:1674 –1679. http://dx .doi.org/10.1128/AAC.46.6.1674-1679.2002. Pisciotta AV. 1980. Concepts of haemostasis and thrombosis: a study of the coagulation defect in hemophilia and in jaundice (Quick, StanleyBrown and Bancroft 1935). Armand J Quick (1894 –1978)—a short biography Thromb Haemost 44:1–5. Bisseling TM, Willems MC, Versleijen MW, Hendriks JC, Vissers RK, Wanten GJ. 2010. Taurolidine lock is highly effective in preventing catheter-related bloodstream infections in patients on home parenteral nutrition: a heparin-controlled prospective trial. Clin Nutr 29:464 – 468. http: //dx.doi.org/10.1016/j.clnu.2009.12.005. Solomon LR, Cheesbrough JS, Bhargava R, Mitsides N, Heap M, Green G, Diggle P. 2012. Observational study of need for thrombolytic therapy and incidence of bacteremia using taurolidine-citrate-heparin, taurolidine-citrate and heparin catheter locks in patients treated with hemodialysis. Semin Dial 25:233–238. http://dx.doi.org/10.1111/j.1525-139X.2011 .00951.x. Shantsila E, Lip GY, Chong BH. 2009. Heparin-induced thrombocytopenia. A contemporary clinical approach to diagnosis and management. Chest 135:1651–1664. http://dx.doi.org/10.1378/chest.08-2830. Shanks RM, Sargent JL, Martinez RM, Graber ML, O’Toole GA. 2006.
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33.
34.
35.
36.
Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces. Nephrol Dial Transplant 21:2247–2255. http://dx.doi.org /10.1093/ndt/gfl170. Green JV, Orsborn KI, Zhang M, Tan QK, Greis KD, Porollo A, Andes DR, Long Lu J, Hostetter MK. 2013. Heparin-binding motifs and biofilm formation by Candida albicans. J Infect Dis 208:1695–1704. http://dx.doi .org/10.1093/infdis/jit391. Shanks RM, Donegan NP, Graber ML, Buckingham SE, Zegans ME, Cheung AL, O’Toole GA. 2005. Heparin stimulates Staphylococcus aureus biofilm formation. Infect Immun 73:4596 – 4606. http://dx.doi.org/10 .1128/IAI.73.8.4596-4606.2005. Handrup MM, Fuursted K, Funch P, Moller JK, Schroder H. 2012. Biofilm formation in long-term central venous catheters in children with cancer: a randomized controlled open-labelled trial of taurolidine versus heparin. APMIS 120:794 – 801. http://dx.doi.org/10.1111/j.1600-0463 .2012.02910.x. Rijnders BJ, Van Wijngaerden E, Peetermans WE. 2002. Catheter-tip colonization as a surrogate end point in clinical studies on catheter-related bloodstream infection: how strong is the evidence? Clin Infect Dis 35: 1053–1058. http://dx.doi.org/10.1086/342905.
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In Vitro Assessment of the Antimicrobial Efficacy of Optimized Nitroglycerin-Citrate-Ethanol as a Nonantibiotic, Antimicrobial Catheter Lock Solution for Prevention of Central Line-Associated Bloodstream Infections Ruth A. Reitzel,a Joel Rosenblatt,a Cheryl Hirsh-Ginsberg,b Kimberly Murray,c Anne-Marie Chaftari,a Ray Hachem,a Issam Raada Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAa; Department of Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAb; School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAc
The rapid, broad-spectrum, biofilm-eradicating activity of the combination of 0.01% nitroglycerin, 7% citrate, and 20% ethanol and its potential as a nonantibiotic, antimicrobial catheter lock solution (ACLS) were previously reported. Here, a nitroglycerincitrate-ethanol (NiCE) ACLS optimized for clinical assessment was developed by reducing the nitroglycerin and citrate concentrations and increasing the ethanol concentration. Biofilm-eradicating activity was sustained when the ethanol concentration was increased from 20 to 22% which fully compensated for reducing the citrate concentration from 7% to 4% as well as the nitroglycerin concentration from 0.01% to 0.0015% or 0.003%. The optimized formulations demonstrated complete and rapid (2 h) eradication of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant enterococci (VRE), multidrug-resistant (MDR) Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, MDR Enterobacter cloacae, MDR Acinetobacter baumannii, MDR Escherichia coli, MDR Stenotrophomonas maltophilia, Candida albicans, and Candida glabrata biofilms. The optimized NiCE lock solutions demonstrated anticoagulant activities comparable to those of heparin lock solutions. NiCE lock solution was significantly more effective than taurolidine-citrate-heparin lock solution in eradicating biofilms of Staphylococcus aureus and Candida glabrata. The optimized, nonantibiotic, heparin-free NiCE lock solution demonstrates rapid broad-spectrum biofilm eradication as well as effective anticoagulant activity, making NiCE a high-quality ACLS candidate for clinical assessment.
C
entral line-associated bloodstream infections (CLABSIs) pose a significant public health problem in the United States. More than 7 million patients require central venous access each year, resulting in an estimated 250,000 CLABSIs, approximately 30,000 attributable deaths (1), and substantial treatment costs of at least $45,000 per infection (2). The majority of CLABSIs occur in chronically catheterized patients, such as cancer, hemodialysis, or critical care patients. Long-term catheters, such as central venous catheters (CVCs), peripherally inserted central catheters (PICCs), ports, and dialysis catheters, are susceptible to internal colonization of the catheter lumens due to repeated catheter manipulation and infusions. Biofilm formation on the catheter surfaces can serve as the primary source of CLABSI and can be highly resistant to eradication by systemic antibiotic therapy. Several bundled measures have been proposed for both treatment and prevention of CLABSIs, most of which center around reducing and preventing colonization at the skin insertion site or at connections (3). While bundles have been shown to decrease the risk of CLABSI in short-term CVCs (⬍30 days of dwell time), approximately 90% of CLABSIs in longer-term catheters are luminally sourced (4, 5). While antimicrobial coatings exist that do cover luminal surfaces, the duration of protection is limited to only a few weeks (6). Because disinfecting antimicrobial catheter lock solutions (ACLSs) can be repetitively instilled, they have emerged as another approach to prevent luminally sourced CLABSIs. Antimicrobial lock therapy solutions for preventing CLABSI have been reported to include prophylactic applications of antibiotics, antifungals, or antiseptics such as ethanol (EtOH) (7, 8). The
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development of microbial resistance is of great concern with prophylactic antibiotic ACLSs (9). Alternatively, high-concentration antiseptic ethanol ACLSs have been studied. An ACLS consisting of 70% ethanol was not effective in decreasing intraluminal CLABSI and was associated with adverse events such as dizziness (10). Ethanol ACLSs with concentrations of greater than 30% have also been associated with clotting, transient dizziness, and compromising catheter integrity in polyurethane catheters (11). Use of ethanol locks with ethanol concentrations greater than 28% has been further advised against due to the significant potential for protein precipitation at the blood-lock solution interface (12). The in vitro biofilm eradication model highly correlated with clinical microbiological eradication from catheter cultures obtained from CLABSI patients that had their catheters locked for 2 h daily with a minocycline-EDTA-ethanol lock solution (13). We reported the efficacy of 0.01% glyceryl trinitrate (GTN; nitroglyc-
Received 4 February 2016 Returned for modification 23 February 2016 Accepted 4 June 2016 Accepted manuscript posted online 13 June 2016 Citation Reitzel RA, Rosenblatt J, Hirsh-Ginsberg C, Murray K, Chaftari A-M, Hachem R, Raad I. 2016. In vitro assessment of the antimicrobial efficacy of optimized nitroglycerin-citrate-ethanol as a nonantibiotic, antimicrobial catheter lock solution for prevention of central line-associated bloodstream infections. Antimicrob Agents Chemother 60:5175–5181. doi:10.1128/AAC.00254-16. Address correspondence to Joel Rosenblatt, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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erin)–7% citrate–20% ethanol as a potential ACLS. Complete eradication of biofilms formed by selected key multidrug-resistant (MDR) organisms within 2 h of exposure were reported, indicating that the combination of nitroglycerin-citrate-ethanol (NiCE) could be a practical candidate for a preventative ACLS (14). In order to optimize a formulation for clinical testing, we adjusted the concentrations of the formulation. In 2000, the U.S. Food and Drug Administration (FDA) issued a warning that concentrated trisodium citrate should not be used as an ACLS due to a reported death from cardiac arrest after a mistaken bolus injection of 46.7% citrate. The FDA further advised that 4% citrate should be used in these settings. Additionally, 4% sodium citrate has been approved for use in blood collection bags for apheresis or blood transfusion where blood/citrate solution is reintroduced into circulation. We reduced the concentration of citrate from 7% to 4% due to these regulatory precedents (15, 16). If the catheter lumen is flushed, the use of nitroglycerin in a lock solution creates the potential for hypotension as a side effect, so minimizing it would be desirable. Although a 0.01% GTN concentration was below bolus doses of nitroglycerin that had been previously used safely (17–20), to further increase safety margins for potential hypotension, we reduced the concentrations of GTN to 0.0015% or 0.003%. The aim of this study was to assess how much the concentration of ethanol would need to be increased to maintain antimicrobial potency as a result of reducing the GTN and citrate concentrations. MATERIALS AND METHODS Bacterial strains. A broad spectrum of highly virulent Gram-positive, Gram-negative, and yeast pathogens from our hospital was used for testing. These strains included methicillin-resistant Staphylococcus aureus (MRSA; strain MDA 120), vancomycin-intermediate Staphylococcus aureus (VISA; MDA 104), methicillin-resistant Staphylococcus epidermidis (MRSE; MDA 146), vancomycin-resistant enterococci (VRE; MDA 119), multidrug-resistant (MDR) Pseudomonas aeruginosa (MDA 118), MDR Klebsiella pneumoniae (MDA 144), MDR Enterobacter cloacae (MDA 134), MDR Acinetobacter baumannii (MDA 133), MDR Escherichia coli (MDA 141), MDR Stenotrophomonas maltophilia (MDA 140), Candida albicans (MDA 117), and Candida glabrata (MDA 115). All pathogens tested were clinical isolates selected from the MD Anderson Infectious Diseases laboratory stock of stored organisms from cancer patient cultures. Fresh organisms were grown on Trypticase soy agar–5% sheep blood (for bacteria) or Sabouraud dextrose agar (for yeast) from glycerol stock overnight at 37°C. For testing, pure culture was inoculated into Muller-Hinton broth (MHB) and diluted to a 0.5 McFarland standard. Additional dilutions were made as necessary for testing. Assessment of antimicrobial efficacy in a biofilm eradication model. In vitro antimicrobial assessments of the optimized NiCE lock solutions were conducted using an established biofilm eradication model, modified from Kuhn et al. (21). Briefly, 1-cm-diameter silicone discs were placed into a 24-well tissue culture plate and incubated with human plasma at 37°C for 24 h. The plasma was then removed, replaced with 1 ml of 5.5 ⫻ 105 CFU of various bacteria and yeast inocula, and incubated at 37°C for an additional 24 h. Inoculum was removed, and the discs were washed with shaking at 100 rpm for 30 min in 0.9% sterile saline in order to remove any planktonic organisms. After the washing step, discs were exposed to 1 ml of various lock solutions and incubated at 37°C for 2 h. The 2-h exposure time was selected because in our hospital 2 h was the longest duration we found that we could reliably remove vascular access lines from use in critically ill cancer patients. Subsequently, discs were removed and placed in 5 ml of 0.9% sterile saline and sonicated (60 Hz and 150 W) for 15 min to disrupt any remaining biofilm. The resulting solutions were quantitatively cultured by plating 100 l onto Trypticase soy agar–5%
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sheep blood for bacterial pathogens or onto Sabouraud dextrose agar for yeast pathogens. All biofilm eradication experiments were performed with six replicates. Optimization of nitroglycerin-citrate-ethanol lock solution. The synergy of the triple combination of agents in the NiCE lock solution was further investigated to determine the optimal concentrations of nitroglycerin and ethanol in the triple combination (nitroglycerin [Baxter, Deerfield IL], citrate [Sigma-Aldrich, St. Louis, MO], ethanol [Sigma-Aldrich, St. Louis, MO]) against MRSA and VRE biofilms. These organisms were selected for this assessment because of the reported resistance of their biofilms to ethanol (22). Citrate was fixed at 4% due to regulatory precedent (15, 16), concentrations of nitroglycerin were reduced to 0.0015% and 0.003%, and ethanol was tested above 20% in 2% increments to determine the lowest concentration of ethanol needed to compensate for the reduction in nitroglycerin. Our previous study (14) showed that at a 20% ethanol concentration, a nitroglycerin concentration of 0.005% was unable to fully eradicate MRSA biofilm in 2 h (14). Hence, the first incremental step increase in ethanol concentration tested here was 22% ethanol. After optimization, NiCE combinations (0.0015% or 0.003% nitroglycerin plus 4% citrate and 22% ethanol) were compared to the previously published 0.01% nitroglycerin–7% citrate–20% ethanol lock solution in the biofilm eradication model against a broad spectrum of CLABSI pathogens. Discs exposed to Mueller-Hinton broth (MHB) were used as positive controls. Additionally, both concentrations of NiCE lock were tested for efficacy against planktonic bacteria in a broth dilution model. Briefly, fresh MRSA, P. aeruginosa, and C. albicans inocula at a 0.5 McFarland standard were diluted to 1.0 ⫻ 106 CFU/ml in MHB. A total of 500 l of each inoculum was exposed for 2 h to 500 l of NiCE lock with concentrations adjusted so that the 1-ml total volume resulted in 0.0015% or 0.003% nitroglycerin– 4% citrate–22% ethanol. After exposure, 100 l was serially diluted and quantitatively cultured for growth on Trypticase soy agar–5% sheep blood (bacteria) or Sabouraud dextrose agar (yeast). Plates were incubated for 24 h at 37°C and enumerated for growth. All solutions were tested in triplicate; MHB was tested as a control solution. Antimicrobial efficacy of optimized NiCE solution compared to taurolidine-citrate-heparin. The antimicrobial efficacy of the optimized NiCE lock solution was compared in the in vitro model to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin to further assess its suitability for clinical testing. Taurolidine-citrate-heparin lock was chosen as a comparator due to an extensive body of clinical trial literature (23, 24) that would enable conclusions to be inferred about comparative clinical efficacy of NiCE lock solution. Taurolidine-citrate-heparin lock solution was prepared from the individual components (taurolidine [Santa Cruz Biotechnology, Dallas, TX], citrate [Sigma-Aldrich, St. Louis, MO], heparin [Fresenius Kabi USA, Lake Zurich, IL]), resulting in the 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin triple combination lock solution. In these experiments optimized NiCE and taurolidine-citrate-heparin lock solutions as well as MHB controls were tested against a broad spectrum of CLABSI pathogens, previously listed above. Antimicrobial efficacy of optimized NiCE lock solution in a luminal biofilm model. To examine the antimicrobial efficacy of optimized NiCE lock as an ACLS, we examined the efficacy of NiCE lock in a modified luminal biofilm model (25–27). Inocula containing a 0.5 McFarland standard of MRSA, P. aeruginosa, VRE, and C. albicans were diluted to 1.0 ⫻ 106 CFU/ml in Muller-Hinton broth (BD, Sparks, MD) and locked with the appropriate priming volume (approximately 0.5 ml) in each lumen of a 5-French double-lumen peripherally inserted central catheter (PICC) (PowerPICC; Bard Access Systems, Salt Lake City, UT). In addition to locking the catheter with extension line clamps, the distal ends of all catheters were clamped to seal. Catheters were incubated for 24 h at 37°C. The inoculum was subsequently drained from each lumen, and the lumens were flushed with 0.9% saline to remove any unattached organisms. Either 0.0015% or 0.003% NiCE lock solution was instilled into each of the
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FIG 1 Synergy of optimized NiCE lock with 0.0015% or 0.003% GTN– 4% citrate–22% ethanol. For both MRSA (MDA 120) and VRE (MDA 119), the triple combination of optimized NiCE lock shows complete eradication of bacterial biofilm within 2 h of exposure. Individual components showed limited eradication within 2 h of exposures. The binary 22% ethanol– 4% citrate combination showed limited eradication of the MRSA biofilm within a 2-h exposure. This indicates that nitroglycerin, citrate, and ethanol interact synergistically in enhancing MRSA biofilm eradication. Results with nitroglycerin-citrate binary combinations were unchanged from those of MHB controls (data not shown), and results with nitroglycerin-ethanol binary combinations were unchanged from those with 22% ethanol (data not shown).
catheter lumens, which were clamped to lock at the proximal and distal ends of the catheter, and incubated at 37°C for 2 h. After exposure, all lock solutions were collected from catheters and cultured to determine the viability of organisms within the flushed lock solution. Catheters were then cut into 30-cm segments and sonicated for 15 min in 5 ml of 0.9% sterile saline. After sonication, the resulting solution was vortexed briefly and serially diluted, and 100 l was quantitatively cultured onto Trypticase soy agar–5% sheep blood (for bacteria) and Sabouraud dextrose agar (for yeast). All plates were incubated for 24 h at 37°C and enumerated for growth. Two catheters were tested for each organism for each test solution; each catheter was then cut into three, 3-cm-long segments for testing. Muller-Hinton broth was locked into catheters as a control solution. Assessment of anticoagulation. Prothrombin time (PT) was tested in the Clinical Chemistry Laboratory at MD Anderson according to standard methods (28). NiCE lock solutions as well as saline and 200 units (U)/ml heparin were tested for anticoagulant activity by PT. Heparin at a concentration of 200 U/ml was selected as a control because of its current use in our hospital as standard practice for preventing intraluminal thrombus formation in vascular catheters. PTs for 10 replicates per lock solution were measured. Statistical analyses. Student’s t test was used for all comparisons. Our hypothesis was that the average number of CFU recovered from the NiCE lock would be less than that of all other comparators. Therefore, all tests were one sided, and a P value of less than 0.05 (P ⱕ 0.05) was utilized to determine statistical significance. Statistical analyses were performed using Microsoft Office Excel 2013 (Microsoft, Redmond, WA).
RESULTS
Optimization and assessment of antimicrobial efficacy of optimized NiCE lock solution. Results for individual components of the NiCE lock solution (22% ethanol, 4% citrate, and 0.0015% and 0.003% nitroglycerin) and the binary combination of 22% ethanol– 4% citrate for antimicrobial efficacy against MRSA and VRE biofilms are presented in Fig. 1. Triple combinations of
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0.0015% nitroglycerin– 4% citrate–22% ethanol and 0.003% nitroglycerin– 4% citrate–22% ethanol produced complete biofilm eradication of MRSA and VRE following a 2-h exposure. In comparison, the single components were unable to fully eradicate these biofilms (Fig. 1). The 4% citrate–22% ethanol binary combination was able to fully eradicate the VRE biofilm but was unable to fully eradicate the MRSA biofilm. Both the binary combinations of nitroglycerin-citrate and nitroglycerin-ethanol had no effect on VRE and MRSA biofilms (data not shown). Results presented in Table 1 indicate that an ACLS with 22% ethanol and reduced citrate (from 7% to 4%) and nitroglycerin (from 0.01% to 0.0015% or 0.003%) concentrations did not diminish the NiCE ACLS effectiveness in rapidly eradicating (within 2 h) biofilms for a broad spectrum of multidrug-resistant bacterial and yeast pathogens. The slight increase in ethanol concentration was able to compensate for the substantial decreases in nitroglycerin and citrate concentrations. Further incremental increases in ethanol concentration were not required, and so were not tested. When tested against planktonic organisms (MRSA, P. aeruginosa, and C. albicans), NiCE lock demonstrated complete killing, with no viable organisms recovered for each strain tested following a 2-h exposure. This result was identical to the results seen with biofilm (Table 1). Muller-Hinton broth controls showed no change in the amount of organisms initially inoculated into the model, approximately 1.0 ⫻ 106 CFU. Comparison of antimicrobial activities of NiCE and taurolidine-citrate-heparin lock solutions. Figure 2 presents results for taurolidine-citrate-heparin and NiCE lock solutions for VISA and C. glabrata biofilms. The other organisms were also comparatively tested, and both lock solutions produced complete eradication of those biofilms within 2 h. The ability of NiCE lock solution to rapidly eradicate biofilm was significantly superior
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TABLE 1 Antimicrobial activity of NiCE lock solutions with 0.0015%, 0.003%, and 0.01% nitroglycerin Antimicrobial activity (CFU/disc) of:a Organism
MHB control (CFU/disc)
0.0015% GTN–4% citrate–22% EtOH
0.003% GTN–4% citrate–22% EtOH
0.01% GTN–7% citrate–20% EtOH
MRSA VISA MRSE VRE P. aeruginosa K. pneumoniae E. cloacae A. baumannii E. coli S. maltophilia C. albicans C. glabrata
5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104 5 ⫻ 104
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
a
All organisms tested were allowed to form biofilms and then exposed to NiCE lock solutions for 2 h. They were subsequently quantitatively cultured to determine efficacy of biofilm eradication. Lower concentrations of nitroglycerin and 4% citrate NiCE lock solutions with 22% ethanol showed no difference compared to previously published results with 0.01% GTN–7% citrate–20% ethanol NiCE lock solution.
at either nitroglycerin concentration to that of taurolidinecitrate-heparin lock solution against VISA (P ⫽ 0.004) and C. glabrata (P ⫽ 0.04) biofilms and was similarly effective against the other pathogens. Antimicrobial efficacy of optimized NiCE lock solution in a luminal biofilm model. Efficacy of NiCE lock after a 2-h exposure to a microbial biofilm in the lumen of a 5-French double-lumen PowerPICC catheter is presented in Fig. 3. For all organisms tested (MRSA, P. aeruginosa, VRE, and C. albicans), both 0.0015% and 0.003% NiCE lock solutions showed significant efficacies compared to the efficacy of the control (P ⬍ 0.05 for the organism tested) in fully eradicating a luminal biofilm within 2 h of exposure to lock (Fig. 3). Additionally, there were no viable organisms cultured from flushed NiCE lock solution after a 2-h lock, in con-
trast to ⬎1 ⫻ 104 CFU cultured from the MHB control lock. This indicates that NiCE lock kills organisms in luminal biofilm rather than simply dispersing live organisms from the biofilm into solution. Assessment of anticoagulation. PT testing showed that the NiCE lock was comparable to 200 IU/ml heparin in inhibiting blood clotting. All replicates for 200 IU/ml heparin, 0.003% NiCE lock, and 0.0015% NiCE lock inhibited clotting for ⬎70 s (the upper limit of assay). Clotting times for the saline controls ranged from 23 s to 24.2 s (Table 2). DISCUSSION
Reducing the nitroglycerin concentration in NiCE lock solutions by 70 to 85% provides an additional margin of safety against tran-
FIG 2 Comparison of the antimicrobial efficacies of optimized NiCE lock to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin. Both 0.0015% and 0.003% NiCE lock solutions show significantly greater eradication of biofilm (within 2 h) than 1.35% taurolidine–3.5% citrate-1,000 U/ml heparin lock solution for both VISA (MDA 104) (P ⫽ 0.004), and C. glabrata (MDA 115) (P ⫽ 0.04). NiCE lock and taurolidine-citrate-heparin lock showed equivalent antimicrobial efficacies for all other organisms tested.
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FIG 3 Antimicrobial efficacy of NiCE lock in a luminal biofilm model. Both 0.0015% and 0.003% NiCE lock solutions fully eradicated MRSA, P. aeruginosa, VRE, and Candida luminal biofilms within 2 h of locking. Control catheters were locked with Muller-Hinton broth.
sient hypotension when lumens are flushed. To compensate for the reduced nitroglycerin concentration, only a slight increase of 2% in ethanol concentration (from 20 to 22%) was needed to sustain antimicrobial efficacy against the most significant CLABSI pathogens. The 22% ethanol concentration is still lower than ethanol concentrations previously tested clinically and below concentrations (⬎30%) that have been implicated in transient adverse events and catheter dysfunction (11). The concentration of citrate was reduced to 4% (from 7%) due to regulatory precedent (15, 16). At the 4% citrate concentration, antimicrobial efficacy was retained (Table 1), and anticoagulant properties were comparable to those of 200 IU/ml heparin (Table 2). Taurolidine lock solutions have been evaluated in clinical trials for prevention of CLABSI, with mixed clinical results. Some trials reported significant decreases in CLABSI versus results with hep-
TABLE 2 Assessment of anticoagulant properties for optimized NiCE lock by prothrombin time measurement PT of lock solution (s)a Replicate Saline 200 U/ml 0.0015% GTN–4% 0.003% GTN–4% no. concn (%) heparin citrate–22% EtOH citrate–22% EtOH 1 2 3 4 5 6 7 8 9 10
23.5 23.0 23.8 23.2 24.2 23 23.6 23.1 23.6 23.4
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70 ⬎70
a Both 0.0015% and 0.003% optimized NiCE lock solutions gave ⬎70 s of clotting time (the assay limit), which was the same result as that with 200 U/ml heparin. The heparin lock concentration of 200 U/ml is normally used in our hospital for maintaining catheter patency. This result supports the idea that the 4% citrate contained in the NiCE lock provides sufficient anticoagulant activity to prevent clotting in the catheter lumens during locking intervals. PT, prothrombin time.
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arin locks (29), and others did not (23). In a multicenter, doubleblind, randomized, controlled clinical trial in hemodialysis patients, Solomon et al. (23) reported that 1.35% taurolidine–3.5% citrate (TC) lock solution did not significantly decrease bacteremia compared to the level in catheters locked with heparin (23). Catheters locked with taurolidine-citrate also had problems with clotting and showed a significant increase in the need for thrombolytic therapy (P ⫽ 0.008). In a subsequent trial, Solomon et al. (30) added heparin to taurolidine-citrate and evaluated 1.35% taurolidine–3.5% citrate–500 U/ml heparin in 106 hemodialysis patients. This addition of heparin significantly increased time to first use of thrombolytics (hazard ratio of taurolidine-citrate-heparin to taurolidine-citrate, 0.2; 95% confidence interval [CI], 0.06 to 0.5) with a nonsignificant change in time to bacteremia (hazard ratio of taurolidine-citrate-heparin to heparin, 0.4; 95% CI, 0.2 to 1.0) (30). The addition of heparin to a lock solution has two significant disadvantages. One is the significant risk of heparin allergy and heparin-associated thrombocytopenia, which reportedly can occur in 10 to 30% of patients receiving heparin (31). A second is that heparin has been shown to promote the formation of staphylococcal (32) and Candida (33) biofilms. Staphylococcal biofilm was promoted by heparin in a dose-dependent manner (34). This is particularly relevant because of the high concentrations of heparin in taurolidine-citrate-heparin required to overcome the prothrombotic tendencies of taurolidine-citrate. A clinical trial conducted in a pediatric cancer population evaluated taurolidine-citrate-heparin versus heparin (35). This study reported a significant reduction in catheter-related bloodstream infections (CRBSIs) with the use of taurolidine-citrate-heparin (0.1 CRBSIs per 1,000 CVC days with taurolidine-citrate-heparin versus 0.9 CRBSIs per 1,000 CVC days with heparin; P ⫽ 0.03); however, while taurolidine-citrate-heparin reduced the rate of CRBSI in this study, intraluminal microbial colonization was detected in most of the taurolidine-citrate-heparinlocked catheters (35). In fact, over 88% of the taurolidine-citrateheparin-locked catheters were colonized with biofilms (23 of 26
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catheters) covering a mean of 80% of the catheter surface. There was no significant difference in catheter colonization rates between the heparin and taurolidine-citrate-heparin groups. In our in vitro study, we compared optimized NiCE lock solutions (0.0015 to 0.003% GTN– 4% citrate–22% EtOH) to 1.35% taurolidine–3.5% citrate–1,000 U/ml heparin for their abilities to eradicate biofilm formed on silicone surfaces. For the majority of the common CLABSI pathogens tested, the NiCE and taurolidinecitrate-heparin lock solutions performed comparably. However, the optimized NiCE lock was superior to taurolidine-citrate-heparin against vancomycin-intermediate Staphylococcus aureus and Candida glabrata biofilms, such that taurolidine-citrate-heparin failed to eradicate these organisms while the NiCE lock achieved complete eradication (Fig. 2). Since catheter colonization is reportedly a prelude to infection (36), NiCE lock therefore has the potential to be similar to or more clinically effective than taurolidine-citrate-heparin in preventing CLABSI. The optimized NiCE lock solutions had anticoagulant properties comparable to those of heparin without the disadvantages of containing heparin. Future planned studies include a human clinical trial assessing the safety and effectiveness of the optimized NiCE lock solutions in catheterized cancer patients. ACKNOWLEDGMENTS We thank Hewan Gebreselassie and Bette Muthomi at The MD Anderson Cancer Center, School of Health Professions, for their assistance with anticoagulation testing.
REFERENCES 1. Klevens RM, Edwards JR, Richards CL, Jr, Horan TC, Gaynes RP, Pollock DA, Cardo DM. 2007. Estimating health care-associated infections and deaths in U.S. hospitals, 2002. Public Health Rep 122:160 –166. 2. Warren DK, Quadir WW, Hollenbeak CS, Elward AM, Cox MJ, Fraser VJ. 2006. Attributable cost of catheter-associated bloodstream infections among intensive care patients in a nonteaching hospital. Crit Care Med 34:2084 – 2089. http://dx.doi.org/10.1097/01.CCM.0000227648.15804.2D. 3. Frasca D, Dahyot-Fizelier C, Mimoz O. 2010. Prevention of central venous catheter-related infection in the intensive care unit. Crit Care 14: 212. http://dx.doi.org/10.1186/cc8853. 4. Raad I, Chaftari AM. 2014. Advances in prevention and management of central line-associated bloodstream infections in patients with cancer. Clin Infect Dis 59(Suppl 5):S340 –S343. http://dx.doi.org/10.1093/cid /ciu670. 5. Mermel LA, Allon M, Bouza E, Craven DE, Flynn P, O’Grady NP, Raad II, Rijnders BJ, Sherertz RJ, Warren DK. 2009. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America. Clin Infect Dis 49:1– 45. http://dx.doi.org/10.1086/599376. 6. Raad I, Reitzel R, Jiang Y, Chemaly RF, Dvorak T, Hachem R. 2008. Anti-adherence activity and antimicrobial durability of anti-infectivecoated catheters against multidrug-resistant bacteria. J Antimicrob Chemother 62:746 –750. http://dx.doi.org/10.1093/jac/dkn281. 7. Maiefski M, Rupp ME, Hermsen ED. 2009. Ethanol lock technique: review of the literature. Infect Control Hosp Epidemiol 30:1096 –1108. http://dx.doi.org/10.1086/606162. 8. Segarra-Newnham M, Martin-Cooper EM. 2005. Antibiotic lock technique: a review of the literature. Ann Pharmacother 39:311–318. http://dx .doi.org/10.1345/aph.1E316. 9. Landry DL, Braden GL, Gobeille SL, Haessler SD, Vaidya CK, Sweet SJ. 2010. Emergence of gentamicin-resistant bacteremia in hemodialysis patients receiving gentamicin lock catheter prophylaxis. Clin J Am Soc Nephrol 5:1799 –1804. http://dx.doi.org/10.2215/CJN.01270210. 10. Slobbe L, Doorduijn JK, Lugtenburg PJ, El Barzouhi A, Boersma E, van Leeuwen WB, Rijnders BJ. 2010. Prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: a randomized, placebo-controlled trial. PLoS One 5:e10840. http://dx.doi.org /10.1371/journal.pone.0010840.
5180
aac.asm.org
11. Mermel LA, Alang N. 2014. Adverse effects associated with ethanol catheter lock solutions: a systematic review. J Antimicrob Chemother 69: 2611–2619. http://dx.doi.org/10.1093/jac/dku182. 12. Schilcher G, Schlagenhauf A, Schneditz D, Scharnagl H, Ribitsch W, Krause R, Rosenkranz AR, Stojakovic T, Horina JH. 2013. Ethanol causes protein precipitation—new safety issues for catheter locking techniques. PLoS One 8:e84869. http://dx.doi.org/10.1371/journal.pone .0084869. 13. Raad I, Chaftari AM, Zakhour R, Jordan J, Al Hamal Z, Jiang Y, Yousif A, Garoge K, Mulanovich V, Viola G, Kanj S, Pravinkumar SE, Rosenblatt J, Hachem R. 2015. Successful salvage of central venous catheters in the setting of catheter related or central line-associated bloodstream infections using catheter lock consisting of minocycline, EDTA, and 25% ethanol, abstr 285. Abstr IDWeek 2015, San Diego, CA, 7 to 11 October 2015. Infectious Diseases Society of America, Arlington, VA. 14. Rosenblatt J, Reitzel R, Dvorak T, Jiang Y, Hachem RY, Raad II. 2013. Glyceryl trinitrate complements citrate and ethanol in a novel antimicrobial catheter lock solution to eradicate biofilm organisms. Antimicrob Agents Chemother 57:3555–3560. http://dx.doi.org/10 .1128/AAC.00229-13. 15. Food and Drug Administration. 2000. Anticoagulant sodium citrate 4% w/v solution, U.S.P.—summary basis of approval. Food and Drug Administration, Silver Spring, MD. http://www.fda.gov/BiologicsBloodVaccines /BloodBloodProducts/ApprovedProducts/NewDrugApplicationsNDAs /ucm082845.htm. 16. U.S. Food and Drug Administration. 2000. FDA issues warning on tricitrasol dialysis catheter anticoagulant. FDA talk paper T00-16. U.S. Department of Health and Human Services, Rockville, MD. 17. Mercier FJ, Dounas M, Bouaziz H, Lhuissier C, Benhamou D. 1997. Intravenous nitroglycerin to relieve intrapartum fetal distress related to uterine hyperactivity: a prospective observational study. Anesth Analg 84: 1117–1120. http://dx.doi.org/10.1097/00000539-199705000-00030. 18. Chedraui PA, Insuasti DF. 2003. Intravenous nitroglycerin in the management of retained placenta. Gynecol Obstet Invest 56:61– 64. http://dx .doi.org/10.1159/000072734. 19. Kurz DJ, Naegeli B, Bertel O. 2000. A double-blind, randomized study of the effect of immediate intravenous nitroglycerin on the incidence of postprocedural chest pain and minor myocardial necrosis after elective coronary stenting. Am Heart J 139:35– 43. http://dx.doi.org/10.1016/S0002 -8703(00)90306-5. 20. Nashed AH, Allegra JR, Larsen S, Horowitz M. 1994. Bolus i.v. nitroglycerin treatment of ischemic chest pain in the ED. Am J Emerg Med 12:288 –291. http://dx.doi.org/10.1016/0735-6757(94)90140-6. 21. Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. 2002. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 46:1773–1780. http://dx.doi.org/10.1128/AAC.46.6.1773-1780 .2002. 22. Redelman CV, Maduakolam C, Anderson GG. 2012. Alcohol treatment enhances Staphylococcus aureus biofilm development. FEMS Immunol Med Microbiol 66:411– 418. http://dx.doi.org/10.1111/1574-695X.12005. 23. Solomon LR, Cheesbrough JS, Ebah L, Al-Sayed T, Heap M, Millband N, Waterhouse D, Mitra S, Curry A, Saxena R, Bhat R, Schulz M, Diggle P. 2010. A randomized double-blind controlled trial of taurolidine-citrate catheter locks for the prevention of bacteremia in patients treated with hemodialysis. Am J Kidney Dis 55:1060 –1068. http://dx.doi .org/10.1053/j.ajkd.2009.11.025. 24. Simon A, Ammann RA, Wiszniewsky G, Bode U, Fleischhack G, Besuden MM. 2008. Taurolidine-citrate lock solution (TauroLock) significantly reduces CVAD-associated grampositive infections in pediatric cancer patients. BMC Infect Dis 8:102. http://dx.doi.org/10.1186/1471-2334 -8-102. 25. Luther MK, Mermel LA, LaPlante KL. 2016. Comparison of telavancin and vancomycin lock solutions in eradication of biofilm-producing staphylococci and enterococci from central venous catheters. Am J Health Syst Pharm 73:315–321. http://dx.doi.org/10.2146/ajhp150029. 26. LaPlante KL, Mermel LA. 2007. In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model. Nephrol Dial Transplant 22:2239 –2246. http://dx.doi.org /10.1093/ndt/gfm141. 27. Shah CB, Mittelman MW, Costerton JW, Parenteau S, Pelak M, Arsenault R, Mermel LA. 2002. Antimicrobial activity of a novel catheter
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Optimized Nitroglycerin-Citrate-Ethanol and CLABSI
28.
29.
30.
31. 32.
lock solution. Antimicrob Agents Chemother 46:1674 –1679. http://dx .doi.org/10.1128/AAC.46.6.1674-1679.2002. Pisciotta AV. 1980. Concepts of haemostasis and thrombosis: a study of the coagulation defect in hemophilia and in jaundice (Quick, StanleyBrown and Bancroft 1935). Armand J Quick (1894 –1978)—a short biography Thromb Haemost 44:1–5. Bisseling TM, Willems MC, Versleijen MW, Hendriks JC, Vissers RK, Wanten GJ. 2010. Taurolidine lock is highly effective in preventing catheter-related bloodstream infections in patients on home parenteral nutrition: a heparin-controlled prospective trial. Clin Nutr 29:464 – 468. http: //dx.doi.org/10.1016/j.clnu.2009.12.005. Solomon LR, Cheesbrough JS, Bhargava R, Mitsides N, Heap M, Green G, Diggle P. 2012. Observational study of need for thrombolytic therapy and incidence of bacteremia using taurolidine-citrate-heparin, taurolidine-citrate and heparin catheter locks in patients treated with hemodialysis. Semin Dial 25:233–238. http://dx.doi.org/10.1111/j.1525-139X.2011 .00951.x. Shantsila E, Lip GY, Chong BH. 2009. Heparin-induced thrombocytopenia. A contemporary clinical approach to diagnosis and management. Chest 135:1651–1664. http://dx.doi.org/10.1378/chest.08-2830. Shanks RM, Sargent JL, Martinez RM, Graber ML, O’Toole GA. 2006.
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33.
34.
35.
36.
Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces. Nephrol Dial Transplant 21:2247–2255. http://dx.doi.org /10.1093/ndt/gfl170. Green JV, Orsborn KI, Zhang M, Tan QK, Greis KD, Porollo A, Andes DR, Long Lu J, Hostetter MK. 2013. Heparin-binding motifs and biofilm formation by Candida albicans. J Infect Dis 208:1695–1704. http://dx.doi .org/10.1093/infdis/jit391. Shanks RM, Donegan NP, Graber ML, Buckingham SE, Zegans ME, Cheung AL, O’Toole GA. 2005. Heparin stimulates Staphylococcus aureus biofilm formation. Infect Immun 73:4596 – 4606. http://dx.doi.org/10 .1128/IAI.73.8.4596-4606.2005. Handrup MM, Fuursted K, Funch P, Moller JK, Schroder H. 2012. Biofilm formation in long-term central venous catheters in children with cancer: a randomized controlled open-labelled trial of taurolidine versus heparin. APMIS 120:794 – 801. http://dx.doi.org/10.1111/j.1600-0463 .2012.02910.x. Rijnders BJ, Van Wijngaerden E, Peetermans WE. 2002. Catheter-tip colonization as a surrogate end point in clinical studies on catheter-related bloodstream infection: how strong is the evidence? Clin Infect Dis 35: 1053–1058. http://dx.doi.org/10.1086/342905.
Antimicrobial Agents and Chemotherapy
aac.asm.org
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