a Institute for Scientific Evaluation of Naturopathy, University of Cologne, Robert-Koch-strasse 10, 50931 Cologne; b Institute of Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 1921, 50935 Cologne, Germany
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Abstract |
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Introduction |
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Prevention of colonization of polymeric surfaces by continuous release of antimicrobial substances incorporated into polymers has been tried as a promising new approach.511 Conflicting clinical results have led to doubts regarding the safety of chlorhexidinesilver sulphadiazine (CSS)-coated catheters.5,1216 Silver may be an ideal candidate for coating devices, as free silver ion concentrations are bactericidal, but silver ions are toxic to human cell cultures.17 As a further development of silicone catheters loaded with rifampicin combinations that have been shown to be colonization and infection resistant to staphylococci in vitro and in vivo,1820 we investigated the release of a highly effective and biocompatible combination of miconazole with rifampicin from a polyurethane catheter. Rifampicin is a competitive inhibitor of bacterial RNA polymerase with potent activity against Gram-positive microorganisms and a high physico-chemical compatibility with hydrophobic polyurethanes. Miconazole is a synthetic antifungal with a wide spectrum of antimicrobial activity and low toxicity and has been used for years in the topical and systemic treatment of mycotic infections. We report on the antimicrobial in vitro efficacy of the miconazolerifampicin-coated catheter against 158 clinical strains isolated from infected CVCs.
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Materials and methods |
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Antibiotic bonding of catheters
Incorporation of the antibiotics into the polymer matrix (1.2% (wt) rifampicin, 5% (wt) miconazole) was performed by a diffusion-controlled process.19,22 The solubility parameters (cohesion energy, cohesion density, Hansen parameter) of each drug were used to determine the most useful information for the compatibility of the polymer with the drugs. These parameters were calculated by reduced solubility and increments of molar attraction contributions as described recently.20,22
Levels of antibiotics in catheter material
Segments of catheters (1 cm) loaded with miconazole rifampicin were extracted in 5 mL of ethyl acetate. The segments were sonicated in ethyl acetate for 15 min. This procedure was repeated three times. After removal of catheter segments the extracted solution was air dried and the retained antimicrobials were suspended in 1 mL of the buffer used for HPLC. Samples of the extracted antibiotic suspension were injected into the HPLC system. Both miconazole and rifampicin were detected under the same conditions as described below.
Controlled release experiment
To measure the drug release kinetics, 1 cm lengths of triple lumen catheter were eluted in phophate-buffered saline (PBS) at 37°C (acceptor volume 10 mL, pH 7). Constant removal, replacement with fresh solvent (every 3 h in early fractions released, after this daily) and stirring were performed to arrive at the approximate diffusional steady state and nearly perfect sink conditions.20,22 The elutions were investigated by HPLC. A HPLC two-pump system delivering a gradient flow was used (HP 1050). An RP8 reverse phase column (Nucleosil ET 200/8/4 5C8, Macherey and Nagel, Duren, Germany) and a pre-column (Nucleosil ET 200/4 Nucleosil 100/5 C8, Macherey and Nagel) were employed. The mobile phase was monitored with a diode array variable wavelength UV-detector (HP 9000 Series 300, Waldbronn, Germany) and data were received by a microprocessor (HP 9000 Series 300). The mobile phase consisted of acetonitrile0.01 M potassium phosphate at 22°C. The solvent gradient started at a 3:7 ratio up to 8:2 with a flow rate of 1.5 mL. The internal standard was p-hydroxybenzole acid-n-butyl ester. Pressure in the column varied from 150 to 190 bar. Wavelengths recorded detecting rifampicin were 254 and 260 nm and that for miconazole was 215 nm.
Antimicrobial stability of coated catheters
Catheters coated with miconazolerifampicin were investigated by the modified KirbyBauer technique to Staphylococcus aureus 5aW1136 and Staphylococcus epidermidis RP62A before or after gas sterilization with ethylene oxide. HPLC measurement of the extracted drugs was performed to detect metabolites from the ethylene oxide-treated catheters. Zones of inhibition produced by coated catheters stored for 12 months (at room temperature, protected from light) were compared with those from fresh coated catheters.
In vitro antimicrobial activity of catheters for 158 clinically isolated strains
The antimicrobial activity of the catheters was determined by the modified KirbyBauer Technique:8,11 158 organisms isolated from infected CVCs during 1998 in the Institute of Medical Microbiology of the University of Cologne [S. epidermidis (n = 112), S. aureus (n = 15), Staphylococcus haemolyticus (n = 2), Pseudomonas aeruginosa (n = 8), Enterococcus faecalis (n = 8), Enterobacteriaceae (n = 7), Candida albicans (n = 3), Corynebacterium spp. (n = 3)] were grown independently for 18 h in MuellerHinton broth (MHB, Oxoid, Basingstoke, UK) to concentrations of 5 x 109 cfu/mL. A cotton swab was placed in the suspension and rubbed across the surface of a MuellerHinton agar plate (Oxoid). Segments of catheter (10 mm) were placed vertically on the agar, overlaid with the organisms and incubated overnight at 37°C. Zones of inhibition around the catheter segments were measured.
In vitro antimicrobial half-life
The half-life of antimicrobial activity of coated catheters was determined by immersing several catheter segments in PBS and incubating the fluid at 37°C. Segments were removed for testing at 2, 5, 7, 10, 15 and 21 days. The antimicrobial activity of extracted catheters against S. epidermidis (n = 3), S. aureus (n = 3), enterococci (n = 3), Candida albicans (n = 3) and Escherichia coli (n = 3) at various intervals was determined by the modified Kirby Bauer technique and compared with the baseline zones of inhibition determined on day 1 before immersion in fluid.
Measurement of bacterial adhesion to the antibiotic-containing polyurethanes
Static adhesion assay.
Antibiotic-loaded and unmodified catheter samples were incubated for 2 h in PBS containing the test strain S. aureus 5aW1136 (isolated from an infected implant). At equilibrium of bacterial adhesion (2 h), the contaminated samples were transferred into MHB and incubated for periods of 24, 48 and 72 h at 37°C. After each incubation period, the bacteria were removed from the polymeric surfaces by ultrasonication (Branson sonifier, Fa. Heinemann, Schwäbisch Gemünd, Germany; 90 s 150 W) and the number of detached viable bacteria was determined by a colony count method.
Dynamic adhesion assay (intraluminal assay).
Antibiotic-loaded catheter tubes (20 cm) and unmodified ones (20 cm) were incubated for 2 h intraluminally with PBS containing the test strain S. aureus 5aW1136. At equilibrium of bacterial adhesion (2 h), the contaminated catheter lumina were washed three times with PBS. Afterwards the lumina were continuously flushed with a peristaltic pump (AmershamPharmacia, Freiburg, Germany) with sterile MHB [80 mL/h, filtered with a sterile filter (0.2 µm)] for periods of 24, 48, 72 and 320 h at 37°C. After each incubation period, 1 cm segments were cut from the distal catheter tube and the bacteria were removed by ultrasonication. The number of detached viable bacteria was determined by a colony count method.
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Results |
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The concentration of miconazole and rifampicin in catheters was 5% (w/w) and 1.2% (w/w), respectively. After 21 days of controlled release, 4% (w/w) miconazole and 0.5% (w/w) rifampicin were still found in the polyurethane.
Controlled release of rifampicin and miconazole from the polyurethane catheter
Perfect sink conditions were used to determine controlled release of antibiotics22 (Figure 1). The release rates of both antimicrobials showed two significant periods. The initial release rate (the so-called burst effect) depends on the mass, distribution and solubility of superficially located antimicrobials. For miconazole and rifampicin, the delivery rate was nearly constant after the burst effect in a range of about 35 µg/cm catheter segment. Antimicrobial delivery exceeded 21 days for both rifampicin and miconazole.
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Catheters coated with miconazolerifampicin did not show any significant difference in in vitro activity against S. aureus or S. epidermidis before or after gas-sterilization with ethylene oxide. HPLC revealed no differences between sterilized and non-sterilized catheters and no metabolites were detected in the ethylene oxide-treated catheters. The zones of inhibition of catheters after 12 months were more than 90% of those of a base line stored at room temperature. We could detect no degradation products.
In vitro antimicrobial activity of coated catheters against 158 clinical strains
Catheter segments coated with miconazolerifampicin demonstrated a broad spectrum of activity against bacteria and C. albicans (Table I). The largest zones of inhibition were associated with Gram-positive bacteria. Against S. epidermidis the mean zone of inhibition was 33 mm compared with 16 mm for catheters coated with CSS. Similar results were obtained for other Gram-positive bacteria such as S. aureus, Enterococcus faecalis and Corynebacterium spp. (Table I
). The activity of miconazolerifampicin coated catheters against Gram-negative bacilli was highest against E. coli and lowest against P. aeruginosa. The mean zone of inhibition for C. albicans was 14 mm (Table I
). The activity against C. albicans of miconazolerifampicin-coated catheters was significantly better than that of those coated with CSS.
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In vitro antimicrobial half-life
Segments immersed in PBS at 37°C were tested after 3, 5, 7, 10, 12, 15, 17 and 21 days against S. epidermidis, S. aureus, E. coli, Corynebacterium spp., Gram-negative bacilli and C. albicans by the modified KirbyBauer technique. The antimicrobial half-life measured by the diameter of inhibition zones of catheters coated with miconazole and rifampin exceeded 21 days (Figures 2 and 3).
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Table II depicts the effectiveness of modified polyurethane catheters containing rifampicin and miconazole in the stationary adhesion assay against S. aureus 5aW 1136. Sterility was achieved after 48 h in contrast to the conventional catheters. In a second perfusion experiment, sterility of colonized antimicrobial catheters was achieved for 14 days (Table II
). Similar results were obtained with S. epidermidis KH11 and RP62A (not shown in detail).
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Discussion |
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Several criteria were taken into consideration in selecting the coating agents used in this study. The antibiotics were not the antimicrobial agents of choice for the treatment of bloodstream infections. To minimize the risk of the development of resistance, combinations of antimicrobials are preferred over single agents.5 The antimicrobials should be capable of molecular migration through the polyurethane. Antimicrobials such as aminoglycosides, fosfomycin and ciprofloxacin were excluded as they are not compatible with the polyurethane.24 A further criterion was that the antimicrobial substances should be active against most strains of staphylococci, Enterobacteriaceae and Candida spp. Neither clindamycin, nor ß-lactam antibiotics and fluoroquinolones such as ciprofloxacin or sparfloxacin possess adequate activity against methicillin-resistant staphylococci, a major cause of catheter-associated infections. In comparison, most coagulase-negative staphylococci isolated from infected catheters are susceptible to rifampicin. In our study, of 112 strains of coagulasenegative staphylococci, 106 strains remained susceptible to rifampicin. Enterobacteriaceae in general were susceptible, but at a lower level. For the Gram-positive cocci isolated from the infected catheters, MICs of miconazole were in the range 0.062 mg/L. Miconazole-resistant staphylococci were not detected. Rifampicin and miconazole act synergically against fungi25 and show no antagonistic effects (investigated by killing curves, unpublished data) unlike glycopeptides, penicillins, cephalosporins, aminoglycosides, quinolones, amphotericin B and tetracyclines.
The activities of miconazolerifampicin-impregnated catheters against Gram-positive and Gram-negative bacteria and C. albicans were significantly superior to the activities of CSS-treated catheters as determined by the zones of inhibition (Table I). Sherertz et al.8 showed that coated devices with zones of inhibition >15 mm were highly predictive of in vivo efficacy whereby colonization is prevented. Against Gram-positive cocci, the imidazole rifampicin catheters demonstrated zone sizes >20 mm, whereas inhibition zones for Gram-negative bacteria and fungi were in the range 1020 mm. The observations of Sherertz et al.8 were supported by early results of an in vivo study, in which rifampicin-loaded, contaminated silicone catheters having inhibition zone sizes >>20 mm successfully withstood bacterial colonization without any clinical signs of foreign body infection.18,26
The total amount of antimicrobials in the modified catheters has been found to be orders of magnitudes less than a single therapeutic dose, and this is released slowly over several weeks (Figure 1). For this reason, major organ toxicity such as hepatic dysfunction is unlikely. A theoretical complication is the local accumulation of antimicrobials in tissues adjacent to the catheter, producing local toxicity or irritation. Previous animal toxicological studies have not shown any such local or systemic irritation or toxicity caused by rifampicin.18,27 The long period of antimicrobial slow release was chosen in order to ensure protection over the whole indwelling period of the catheter. Attempts to decrease the concentration of rifampicin led to a failure of protection for the target period.19
Any coated catheter may elicit an allergic or immune response. Following the growing number of anaphylactic reactions in Japan, CSS catheters were banned in 1997.26 In comparison, the substances miconazole and rifampicin are very unlikely to induce mutagenicity or sensitization.28
Development of resistance in the clinical use of antimicrobial-coated catheters is a frequently discussed problem. Resistance could develop at the entry site of all types of catheters. Attempts to induce bacterial mutations [S. epidermidis strains (n = 5)] on gradient plates with miconazolerifampicin combinations failed (results not shown); resistant strains could not be detected in chequerboard experiments or in killing experiments, indicating the ability of the combination to prevent the development of resistance (unpublished data).
In this study, anti-infective catheters coated with miconazolerifampicin had a broad spectrum of in vitro activity against staphylococci, other Gram-positive bacteria, C. albicans and various Gram-negative bacilli. The efficacy of the miconazolerifampicin-coated catheter remained stable at room temperature for more than a year and the half-life of antimicrobial activity exceeded 3 weeks. The in vitro results are therefore encouraging and this antimicrobial polyurethane catheter with a long-lasting efficacy may fulfil the prediction made by Dennis Maki: "Binding of a non-toxic antimicrobial drug to the catheter surface or incorporation of the substance into the catheter material itself may ultimately prove to be the most effective technological innovation for reducing the risk of device-related infections."29
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Notes |
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References |
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Received 20 October 1999; returned 11 November 1999; revised 21 December 1999; accepted 1 February 2000