ABSTRACT
Central-line-associated bloodstream infections are increasingly recognized to be associated with intraluminal microbial biofilms, and effective measures for the prevention and treatment of bloodstream infections remain lacking. This report evaluates a new commercially developed antimicrobial catheter lock solution (ACL), containing trimethoprim (5 mg/ml), ethanol (25%), and calcium EDTA (Ca-EDTA) (3%), for activity against bacterial and fungal biofilms, using in vitro and in vivo (rabbit) catheter biofilm models. Biofilms were formed by bacterial (seven different species, including vancomycin-resistant Enterococcus [VRE]) or fungal (Candida albicans) species on catheter materials. Biofilm formation was evaluated by quantitative culture (CFU) and scanning electron microscopy (SEM). Treatment with ACL inhibited the growth of adhesion-phase biofilms in vitro after 60 min (VRE) or 15 min (all others), while mature biofilms were completely inhibited after exposure for 2 or 4 h, compared to control. Similar results were observed for drug-resistant bacteria. Compared to the heparinized saline controls, ACL lock therapy significantly reduced the catheter bacterial (3.49 ± 0.75 versus 0.03 ± 0.06 log CFU/catheter; P = 0.016) and fungal (2.48 ± 1.60 versus 0.55 ± 1.19 log CFU/catheter segment; P = 0.013) burdens in the catheterized rabbit model. SEM also demonstrated eradication of bacterial and fungal biofilms in vivo on catheters exposed to ACL, while vigorous biofilms were observed on untreated control catheters. Our results demonstrated that ACL was efficacious against both adhesion-phase and mature biofilms formed by bacteria and fungi in vitro and in vivo.
INTRODUCTION
Central venous catheters (CVCs) are intravascular devices that are inserted centrally or peripherally and terminate at the heart or in a proximal great vessel. CVCs are indispensable tools in acute hospital care and outpatient settings; they enable infusion therapy, such as fluid management, intravenous administration of drugs, cancer chemotherapy, hemodynamic monitoring, administration of parenteral nutrition, and hemodialysis. CVCs provide continuous access to the central venous system without the need for repeated venipuncture (1).
All intravascular catheters communicate with the vascular system and therefore must be filled with a physiologically compatible solution to create a hydrostatic lock between the catheter hub and the circulatory system. Lock solutions physically oppose the retrograde flow of blood back into the indwelling catheter; in doing so, however, they support microbial growth and biofilm formation inside catheters that are isolated from immune surveillance. Biofilms are complex three-dimensional structures composed of microorganisms living in a microbe-generated extracellular matrix of proteins, nucleic acids, and polysaccharides, and they are characteristically less susceptible to antimicrobial agents (2, 3). Biofilms formed by bacteria (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter spp.) and fungi (e.g., Candida albicans) have the potential to go through cycles of active shedding, thus creating a nidus of systemic pathogen dissemination that may be the cause of recurring bloodstream infections (BSIs) and sepsis (2–9).
Globally, the standard of care in lock solutions is phosphate-buffered saline (PBS) or PBS containing heparin. Heparin is a polysaccharide that is used as an anticoagulant to prevent blood coagulation within the catheter lumen (10). Although heparin is a potent anticoagulant, it is associated with BSIs and bleeding complications (11).
Chronically or critically ill patients with indwelling CVCs are at risk of developing central-line-associated bloodstream infections (CLABSIs), which contribute to patient morbidity, patient deaths, extended hospital stays, and increased costs of care (12–17). According to the Agency for Healthcare Research and Quality in the U.S. Department of Health and Human Services, CLABSIs are responsible for an estimated 84,551 to 203,916 preventable infections each year, with costs of $1.7 to $21.4 billion (18). Mortality rates for diagnosed CLABSIs range from 12 to 25%, according to the National Healthcare Safety Network (19).
In recent years, several antimicrobial catheter lock solutions (ACLs) have been described and clinically evaluated. Meta-analyses of human clinical trials involving off-label compounded antibiotic lock solutions have demonstrated statistically significant reductions in catheter-associated BSIs (20), as have studies of taurolidine-containing lock solutions (21).
In this report, we describe a new commercially prepared ACL containing the antibiotic trimethoprim (TMP) at 5 mg/ml, 25% (vol/vol) ethanol, and 3% Ca-EDTA in a physiologically balanced PBS solution (22, 23). This study evaluated the activity of ACL against early-adhesion-phase and 24-h-mature-phase bacterial and fungal biofilms, using in vitro and in vivo (rabbit) catheter biofilm models. The data demonstrate that ACL is efficacious in both preventing biofilm formation (adhesion phase) and acting against mature biofilms formed by bacteria and fungi.
RESULTS
Activity of ACL against catheter-associated biofilms in vitro.The data demonstrated that ACL was able to prevent biofilm formation in the adhesion phase by resistant and susceptible Escherichia coli, Enterococcus faecium, and P. aeruginosa strains, as well as methicillin-resistant S. aureus (MRSA) and C. albicans strains, after 15 min of exposure (log CFU of 0 for all organisms) (Fig. 1). ACL was also able to prevent biofilm formation by resistant Enterobacter cloacae (n = 1) and Serratia marcescens (n = 1) after 15 min of exposure (log CFU of 0). Vancomycin-resistant Enterococcus (VRE) biofilms were the most resistant of the strains tested. ACL treatment of VRE discs resulted in a 1.75-log-unit reduction of CFU, compared to PBS-treated control discs, at 15 min and a 3.09-log-unit reduction of CFU at 30 min of exposure (log CFU values of 1.32 and 0 versus 3.07 and 3.09, respectively). Increasing the ACL exposure time for VRE biofilms to 60 min resulted in complete eradication and no outgrowth (log CFU of 0).
Log CFU values (average ± SD) for ACL-treated disks and untreated control disks against adhesion-phase biofilms formed by TMP-susceptible and TMP/multidrug-resistant E. coli (n = 3), E. faecium (n = 2), P. aeruginosa (n = 3), MRSA (n = 4), and C. albicans (n = 3).
Exposure of mature biofilms formed by resistant and susceptible E. coli, E. faecium, and P. aeruginosa strains, as well as MRSA and C. albicans strains, to ACL for 2 or 4 h also resulted in the eradication of biofilms, compared to PBS-treated controls, with ACL-treated disks demonstrating log CFU values of 0, while the PBS-treated control disks yielded average log CFU values of 5.25 to 6.73 (Fig. 2). Against 1 strain each of resistant E. cloacae, S. marcescens, and VRE, ACL was also able to eliminate mature biofilms after 2 and 4 h of exposure. The ACL-treated disks all had log CFU values of 0, while the PBS-treated control disks had log CFU values of 4.43 to 6.15.
Log CFU values (average ± SD) for ACL-treated disks and untreated control disks against mature biofilms formed by E. coli (n = 3), E. faecium (n = 2), P. aeruginosa (n = 3), MRSA (n = 4), and C. albicans (n = 3).
Efficacy of ACL against catheter-associated biofilms in vivo.Next, we evaluated the in vivo efficacy of ACL against bacterial and fungal biofilms. For evaluation of efficacy against bacterial biofilms, blood samples obtained from rabbit model catheters on day 3 postinoculation (prior to the initiation of lock therapy) were positive for MRSA; this confirmed the presence of catheter-associated MRSA biofilm infections. Untreated controls yielded an average bacterial burden of 3.49 ± 0.75 log CFU/catheter (Fig. 3A). In contrast, ACL-treated catheters yielded an average bacterial burden of 0.03 ± 0.06 log CFU/catheter (P = 0.016) (Fig. 3A). Lock therapy with ACL completely cleared MRSA infections in 3 of 4 catheters, yielding log CFU values of 0 in each (the fourth catheter had a log CFU value of 0.13). Scanning electron microscopy (SEM) showed eradication of biofilms from catheters exposed to ACL, while heavy biofilms with typical structural and architectural features were observed in untreated control catheters (Fig. 3B and C).
(A) Box plot of bacterial burdens of catheters obtained from ACL-treated animals and untreated control animals inoculated with MRSA biofilms in vivo. The shaded area represents data between the first quartile and the third quartile, the black lines show the median values, and the bars show the minimum and maximum data points. (B and C) Representative scanning electron micrographs of the intraluminal surface of catheters obtained from ACL-treated animals (B) and untreated animals (C). Magnification, ×5,000.
For evaluation of antifungal biofilm efficacy, blood culture samples obtained from catheters on day 3 postinfection (prior to the initiation of lock therapy) were positive for C. albicans, again confirming the presence of catheter-associated biofilm infections. There was a significant difference in the mean log CFU values between animals treated with ACL (0.55 ± 1.19 log CFU/catheter segment) and untreated controls (2.48 ± 1.60 log CFU/catheter segment; P = 0.013) (Fig. 4). Lock therapy with ACL completely cleared fungal cells in 8 catheters (log CFU values of 0 for each).
Box plot of fungal burdens of catheters obtained from ACL-treated animals and untreated control animals inoculated with C. albicans biofilms in vivo. The shaded area represents data between the first quartile and the third quartile, the black lines show the median values, and the bars show the minimum and maximum data points. O2, outlier.
DISCUSSION
These studies demonstrated that ACL successfully prevented formation of biofilms and also inhibited mature biofilms formed by multidrug-resistant bacteria and fungal species known to cause catheter-associated BSIs in humans. Previous studies reported variable antimicrobial activities of ethanol and EDTA, alone or in combination. In this regard, Passerini de Rossi et al. (24) showed that exposure of mature biofilms formed on silicone catheters to ethanol (25 or 40%) or a combination of ethanol (25%) and EDTA (30 mg/ml) for 1 h resulted in inhibition of biofilm growth. However, complete eradication was not observed for EDTA treatment, even after exposure for 24 h (24). In a separate study, Parra et al. (25) reported that antibiotics (daptomycin, teicoplanin, and clarithromycin) combined with ethanol (35%) significantly reduced biofilms formed by methicillin-resistant coagulase-negative staphylococci with 72 h of exposure. These studies are in agreement with our findings that combination treatment with an antibiotic and ethanol in an aqueous solution is effective against mature biofilms formed by Gram-positive bacteria, Gram-negative bacteria, and fungal pathogens.
Although there is increasing recognition that antibiotic lock solutions can reduce the risk of infection, the standard lock solutions used clinically remain PBS alone and PBS containing heparin as an anticoagulant agent (26). Heparin is a sulfated polysaccharide analogous to carrageenan, a sulfated polysaccharide that is isolated from marine algae and is used as a growth substrate in microbiology plates to culture bacteria (10). Moore et al. (27) compared the efficacy of gentamicin-citrate and heparin catheter lock solutions and reported significant reductions (73%) in the rates of BSIs; a limitation of using off-label gentamicin-citrate lock solutions is that, rather than being microbicidal, the combination was only bacteriostatic and resulted in increased recovery of gentamicin-resistant bacteria in the treatment cohort, compared to the heparin cohort. Murray et al. (28) reported that introduction of taurolidine-citrate-heparin resulted in a significant reduction (56%) in staphylococcal bacteremia in hemodialysis patients. Saxena et al. (29) showed that cefotaxime-heparin locks for tunneled cuffed catheters reduced the incidence of catheter-related BSIs associated with methicillin-susceptible S. aureus (MSSA) but did not show activity against MRSA. Overall, these investigations have led to the suggestion that heparin lock solution should be abandoned due to the risks of rapid biofilm growth and bleeding (30).
Results from this study expanded on previous publications by demonstrating that ACL was effective in preventing adhesion-phase cells from developing into mature biofilms and also was effective in eradicating fully formed mature biofilms. Our results also showed that, with 7 days of lock therapy, ACL was able to completely inhibit outgrowth from bacterial and fungal biofilms formed in vivo. This novel lock solution may provide important advantages in preventing intraluminal biofilm formation and thereby preventing other nosocomial infections from being introduced into the bloodstreams of patients with indwelling catheters that must be routinely handled by health care professionals. Furthermore, use of this novel lock solution may help avoid the risk and added expense of removing infected indwelling catheters. Based on these findings, further clinical testing of this novel lock solution is warranted.
MATERIALS AND METHODS
Organisms and reagents.Organisms tested for the catheter-associated biofilm model were Escherichia coli, Enterococcus faecium, Enterobacter cloacae, P. aeruginosa, MRSA, VRE, Serratia marcescens, and C. albicans (Table 1). Bacterial isolates were obtained from the clinical microbiology laboratory (Michael Jacobs) of University Hospitals Cleveland Medical Center, while fungal isolates were obtained from the collection of the Center for Medical Mycology at Case Western Reserve University. The antimicrobial catheter lock solution containing 5 mg/ml TMP, 25% (vol/vol) ethanol, and 3% Ca-EDTA was prepared according to good manufacturing practices by AAI Pharma Services Corp. (now Alcami, Inc.).
Susceptibility profiles for bacterial strains
Rationale for the selection of trimethoprim, ethanol, and Ca-EDTA for the formulation.Our aim was to devise a lock solution that was safe for use in humans, that matched the density, osmolality, and pH of human plasma, that was commercially manufacturable and stable over long periods, and that exerted fast-acting and broad-spectrum microbiocidal activity against established biofilms of Gram-positive bacteria, Gram-negative bacteria, and pathogenic fungi.
(i) Rationale for the selection of ethanol.Ethanol was selected as an excipient to maintain the stability of the solution and to enhance the activity of TMP. Ethanol contributes to the microbiocidal activity of TMP through its ability to permeate and to destabilize microbial membranes. This leads to improvement in the penetration and retention of TMP in the cytoplasm, where TMP inhibits the folate biosynthesis pathway, eventually causing cell death. The optimal concentration of ethanol (25%) was determined experimentally in in vitro studies of MRSA biofilms, using levels that ranged from 10% to 80%. Although 25% (vol/vol) ethanol was effective in eradicating Candida biofilms, it alone was not sufficient to inhibit MRSA biofilms completely; therefore, TMP was included in the formulation. Previous reports demonstrated that alcohol dehydrogenase (ADH) is downregulated in Candida biofilms, and ADH restricts biofilm formation through an ethanol-dependent mechanism (31). Ethanol at concentrations of ≥10% significantly reduced Candida biofilms (40%, compared to untreated controls; P < 0.05) (31). Further testing showed that 25% ethanol was active against mature-phase biofilms formed by C. albicans. Since high concentrations of ethanol might affect the integrity of catheter materials, ethanol was included at a concentration of 25% (vol/vol) in the designed formulation.
(ii) Rationale for the selection of trimethoprim.Since ethanol (25%) showed insufficient in vitro activity against MRSA biofilms, it was decided that an antibacterial agent should be included in our formulation. TMP was selected because it is safe and approved for use in humans, it is highly stable, and it inhibits dihydrofolate reductases from Gram-positive and Gram-negative bacteria with little to no activity on mammalian enzymes, thus offering a very large therapeutic index. Preliminary experiments were conducted to evaluate the antibiofilm activity of TMP alone and combined with ethanol. Initially, TMP alone was evaluated at 1, 5, and 10 mg/ml. Our data showed that TMP alone was not active against biofilms. Subsequently, the activities of these concentrations of TMP combined with 25% ethanol were tested against adhesion-phase (exposure for 15, 30, or 60 min) and mature-phase (exposure for 1 or 2 h) biofilms. Results showed that all combinations were active against both bacterial and fungal biofilms. Therefore, TMP was included at a concentration of 5 mg/ml in the designed formulation.
(iii) Rationale for the selection of Ca-EDTA.Ca-EDTA is used as a stabilizer and preservative in a number of pharmaceutical preparations, at maximum amounts of up to 10% in intravenous formulations (European Pharmacopoeia), and in other types of personal care products, such as contact lens solutions. It is included in the lock solution formulation as a stabilizer and may contribute to the antimicrobial and anticoagulant activities of the combined solution. To select the appropriate concentration of Ca-EDTA to use in the formulation, three concentrations of Ca-EDTA (1, 2, and 3%) were evaluated using the whole-blood clotting time method (32). The results showed that 3% Ca-EDTA, used singly or in combination with ethanol and TMP, prevented the clotting of whole blood for 7 days. In contrast, formulations containing 1% or 2% Ca-EDTA yielded the formation of thick clumps and crystals in the blood by 2 h postexposure. Positive and negative controls behaved as expected; exposure of blood to PBS alone resulted in a solid clot after 38 min, while the mixture of blood and water solidified after 14 min. Therefore, Ca-EDTA was included at a concentration of 3% (wt/vol) in this formulation.
Biofilm formation.Early adhesion- and mature-phase biofilms were formed according to our standardized protocol, as described previously (2, 4). Fungal and bacterial biofilms were grown on silicone elastomer disks with a 1.5-cm diameter (Cardiovascular Instrument Corp., Wakefield, MA). To facilitate biofilm growth and adhesion, sterile disks were placed into 12-well tissue culture plates containing 2 ml of fetal bovine serum (FBS) each. Plates were placed on a rocker and incubated for 24 h at 37°C. Disks were then removed and washed with PBS to eliminate any remaining FBS. For the formation of early (90-min) adhesion-phase biofilms, FBS-coated disks were then soaked in 3 ml of a suspension of 1 × 107 cells/ml and incubated for 90 min at 37°C. After 90 min, wells were gently washed with PBS to remove nonadherent cells. Following adhesion-phase biofilm formation, discs were incubated for 15, 30, or 60 min in the presence of ACL (4 ml) or PBS (control).
For the evaluation of activity against mature biofilms, bacterial and fungal cultures were allowed to establish mature biofilms for an additional 24 h (bacteria) or 48 h (Candida). Mature biofilms of bacteria and Candida were then exposed to 4 ml of PBS (control) or ACL for 2 and 4 h.
Activity of the lock solution against microbial biofilms in vitro.At each time point, aliquots of adhesion- and mature-phase PBS-treated control biofilms and ACL-treated biofilms were harvested. Silicone elastomer discs were then scraped, and the scraped material was suspended in 100 μl. Ten-fold serial dilutions were made and spread on potato-dextrose agar or brain heart infusion agar plates to enable microbial growth and yield CFU. Plates were incubated for 24 h at 37°C, and CFU were counted. Discs incubated with PBS alone were used as controls.
Efficacy of the catheter lock solution in vivo.ACL was also tested against biofilms formed by C. albicans or MRSA in vivo, using our previously described rabbit lock therapy model (33). A standard inoculum (300 μl) consisting of 107 CFU/ml of C. albicans (M61) or MRSA (ATCC 43300) was prepared in sterile normal saline with 100 U of heparin (Abbott Laboratories, North Chicago, IL). Female New Zealand White rabbits weighing 2.5 to 3.5 kg (Covance, Inc., Princeton, NJ) were housed at the Case Western Reserve University Animal Facility, adhering to Institutional Animal Care and Use Committee guidelines. After the animals were allowed to acclimatize for 7 days, surgery was performed to place a silicone catheter in the external jugular vein. The catheters were made with silastic tubing with an internal diameter of 0.04 in. and an external diameter of 0.085 in., cut to 30 cm (Dow Corning, Midland, MI). To protect the catheter from collapse and to ensure that it was properly positioned, a polyethylene cuff (PE 240; Becton Dickinson, Sparks, MD) was slipped over the catheter and attached with cyanoacrylate glue 4 cm from the inserted end. Prior to surgery, rabbits were anesthetized intramuscularly using a cocktail of ketamine and xylazine. The animals were clipped, and a surgical scrub was performed. Silicone catheters were placed in the right external jugular vein and tunneled subcutaneously, as described previously (31). Catheters were inoculated with 107 CFU/ml of C. albicans or MRSA, which was “locked” in the lumen of the catheter for 24 h to allow the formation of intraluminal biofilm. Free-floating organisms were then removed by aspirating the inoculum broth, and daily heparinized saline flushes (100 U of heparin in sterile normal saline) were performed for 3 days. Once mature biofilms were formed, blood samples were obtained from the catheter and submitted for culture to confirm the presence of MRSA or C. albicans. Animals inoculated with MRSA were randomized into the following groups: (i) ACL lock therapy for 2 h/day for 7 days (n = 4) and (ii) untreated control (n = 5). Similarly, animals inoculated with C. albicans were randomized into the following groups: (i) ACL lock therapy for 2 h/day for 7 days (n = 16) and (ii) untreated control (n = 8). Untreated control catheters were flushed daily with heparinized saline. After completion of each daily treatment with ACL, the solution was removed and the ACL-treated catheters were flushed with 300 μl of heparinized saline. The animals were then euthanized with a cardiac injection of pentobarbital, and the catheters were removed for quantitative culture and SEM, as described previously (33).
Quantitative catheter culture.Catheters were cut into 1-cm segments; these segments were cut longitudinally and placed in sterile saline (10 ml). One segment was set aside for SEM analysis. The remaining segments were pooled, sonicated at 40,000 Hz (Bransonic 1510; Branson Ultrasonics Corp., Danbury, CT) for 12 min in 4-min periods, and vortex-mixed for 15 s. Suspensions were serially diluted, and 1-ml aliquots were spread onto plates containing Sabouraud dextrose agar (Difco Laboratories) supplemented with chloramphenicol and gentamicin. Plates were incubated for 48 h at 37°C, and CFU were counted. Catheters resulting in ≥100 CFU were considered infected, based on previous studies (34).
Scanning electron microscopy.Catheter pieces were fixed in 2% glutaraldehyde, dehydrated, sputter-coated with gold-palladium (60:40), and viewed with a Phillips XL30 scanning electron microscope, as described in our previous publications (35).
Statistical evaluation.The mean log CFU values from quantitative catheter cultures were compared with the Mann-Whitney test, using SPSS software (version 24; IBM Corp., Armonk, NY). P values of <0.05 were considered significant.
ACKNOWLEDGMENTS
Funding support from the NIH (grant R01DE024228) to M.A.G. and P.K.M. and from the Steris Foundation (research award to P.K.M.) is acknowledged.
FOOTNOTES
- Received 11 April 2018.
- Returned for modification 23 April 2018.
- Accepted 30 May 2018.
- Accepted manuscript posted online 4 June 2018.
- Copyright © 2018 American Society for Microbiology.