In Vitro and In Vivo Activity of a Novel Catheter Lock Solution against Bacterial and Fungal Biofilms

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.).

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.