In this paper, we describe the first clinically relevant animal model of catheter-related C. albicans
biofilm infection. Using this model, we show that liposomal amphotericin B antifungal lock therapy can sterilize infected catheters. Experiments aimed at optimizing this animal model established by both quantitative culture and SEM analyses that mature biofilms formed equally on polyurethane and silicone CVCs. Previously, Hawser and Douglas reported that C. albicans
biofilm formation in vitro was significantly decreased when grown on polyurethane compared to silicone elastomer disks (12
). Our in vivo studies, however, indicate that there is no difference in C. albicans
biofilm formation between catheters made of polyurethane and those made of silicone. One explanation for this discrepancy between in vitro and in vivo data may be the formation of conditioning films, or fibrin sheaths, on the intraluminal catheter surface after exposure to blood in vivo. Conditioning films, rich in fibronectin and fibrin, have been shown to coat the surfaces of catheters exposed to blood within hours after catheter insertion (20
). Once formed, the conditioning film masks the properties of the underlying catheter material and enhances the adherence of C. albicans
). This finding may explain why there appeared to be no difference between in vivo biofilm formation on silicone and polyurethane catheters.
During the development of this model, we were surprised to find that only 26% of the untreated animals had positive peripheral blood cultures despite documented catheter infection by both quantitative catheter culture and SEM. However, three of five animals with positive peripheral blood cultures had clotted catheters that had been flushed multiple times immediately before the cardiac puncture was performed. The injection of saline through the catheter lumen may have caused a detachment of yeast cells from the catheter-associated biofilms leading to systemic infection. The continuous infusion of fluids through the infected catheters, as routinely occurs in the clinical setting, may lead to a greater percentage of positive peripheral blood cultures. However, in this model, catheters were flushed daily but were otherwise closed to the systemic circulation.
To our knowledge, this study provides the first in vivo SEM images of C. albicans
biofilms adherent to CVC surfaces. Hawser and Douglas, as well as our group, have previously published in vitro SEM images of C. albicans
). The dense network of yeast cells, germ tubes, and hyphal forms that comprised the in vitro biofilms are difficult to distinguish in vivo. Instead, the in vivo biofilms were composed of a dense basal layer of yeast cells adherent to the catheter material encased in a thick extracellular matrix. Similar findings have been reported by our group using confocal scanning laser microscopy to examine C. albicans
biofilms formed on silicone elastomer disks in vitro (6
). It is possible that the dense network of yeast cells, germ tubes, and hyphal forms may exist in vivo, but they are not visible due to the limitation of SEM to visualize only the surface topography of the biofilm.
To document the clinical utility of this animal model, we compared the ability of fluconazole and liposomal amphotericin B antifungal lock solutions to sterilize CVCs infected with C. albicans
biofilms. We selected the antibiotic lock technique because it was developed as a means to overcome the high-grade antimicrobial resistance observed by bacterial biofilms infecting CVCs. By instilling high concentrations of antibiotics into the lumen of infected catheters, the antibiotic lock technique is able to (i) overcome the increased resistance of microorganisms in the biofilm state, (ii) avoid systemic drug toxicity and eliminate the need to check drug levels in serum, and (iii) prevent distant spread of microorganisms by closing the catheter during treatment (5
). Several open trials of antibiotic lock therapy, with or without concomitant systemic antibiotics, have reported successful catheter salvage without relapse in 83% of those infected with bacteria; however, only 30% of catheters with fungal infections have been successfully retained by using the antibiotic lock technique (17
Two case reports have documented the successful salvage of catheters infected with C. albicans
by using amphotericin B deoxycholate antifungal lock therapy. Johnson et al. (14
) salvaged two catheters by instilling 3 ml of a 2-mg/ml amphotericin B deoxycholate solution and allowing it to dwell for 12 h per day for 10 to 14 days. A report of the salvage of two more catheters infected with C. albicans
after treatment with 3 ml of a 2.5-mg/ml amphotericin B deoxycholate solution locked into the catheter lumen for 14 days, with 7 days of concomitant systemic antifungal therapy, was recently published by Viale et al. (24
). These results suggest that antifungal lock therapy may be an effective strategy for the retention of catheters infected with C. albicans.
The chances of successful catheter salvage may be improved if antifungal agents with enhanced antibiofilm activity are used. Recent in vitro studies performed by our group have identified lipid formulations of amphotericin B and echinocandins as having improved antibiofilm activity compared to other antifungal agents (16
). In this study, we validated these findings with our newly developed animal model. Catheters treated with a liposomal amphotericin B antifungal lock solution were completely sterilized, while catheters treated with a fluconazole antifungal lock solution showed no significant difference in C. albicans
CFU compared to untreated controls.
Suboptimal fluconazole concentrations in the lock solution may explain its inability to sterilize the infected catheters. According to previously published in vitro data, the fluconazole and liposomal amphotericin B MICs for mature biofilms grown with C. albicans
strain M61 are >256 and 0.25 μg/ml, respectively (16
). Therefore, the concentration of liposomal amphotericin B antifungal lock solution used in this study was 12,000 times greater than the MIC for biofilm, while the concentration of fluconazole antifungal lock solution was <11 times the MIC for biofilm. Increased concentrations of fluconazole (300 to 3,000 mg in 300 μl) may improve its efficacy as an antifungal lock solution. However, dissolving the higher concentrations of fluconazole into the small volumes needed for the antifungal lock technique proved to be difficult in this study.
Blood cultures drawn through the infected catheters in the antifungal lock experiments revealed some interesting observations. There seemed to be an exceptionally high rate of catheter thrombosis despite allowing heparin to dwell in the catheter lumens at all times. Thirty-three percent of catheters 3 days postinoculation were clotted. Of the cultures drawn at the completion of the treatment period, 57% of catheters were clotted in the untreated control, while only 14% of catheters were clotted in both the liposomal amphotericin B- and fluconazole-treated groups. The relationship between catheter thrombogenicity and catheter-related infection has been well documented in several studies (21
). However, it remains unclear whether catheter thrombosis promotes catheter-related infection or if the infection leads to thrombosis. In this study, more episodes of catheter thrombosis occurred in the untreated control group than the treated groups, suggesting that infection promotes catheter thrombosis.
Despite the complete sterilization of the catheters treated with liposomal amphotericin B by quantitative culture, 2 out of 7 blood cultures drawn from the catheters were positive for yeast. Although SEM of these catheters revealed almost completely clean intraluminal surfaces, each catheter examined had 1 to 2 small patches of C. albicans
surrounded by minimal amounts of damaged matrix. There is a strong possibility that these adherent cells were dead, but their viability could not be determined by our methods. These findings suggest that perhaps extending the treatment period to 10 to 14 days, as in the successful case reports with amphotericin B deoxycholate, may result in the complete eradication of fungal elements from catheters (14
Even though fluconazole lock therapy was unable to sterilize the intraluminal surface of catheters as documented by quantitative catheter culture and SEM, only 1 out of 7 catheter-drawn blood cultures grew yeast at the completion of the treatment period. The fluconazole lock solution may only kill free-floating planktonic C. albicans released from mature biofilm. The remaining yeast cells are encased in a thick extracellular matrix, as documented by SEM, which may not be released with routine blood draws through the catheter. Sonication of the treated catheters causes a disruption of the matrix with the release of yeast that is isolated by quantitative culture. Therefore, fluconazole lock therapy may represent an effective suppressive therapy for preventing continuous seeding of the bloodstream despite not sterilizing the catheter surface. A possible drawback to such a treatment strategy is the return of infection once the suppressive agent is stopped.
In summary, we describe the development of the first animal model of catheter-related C. albicans biofilm infection and show that this model has utility in preclinical evaluation of antifungal agents and in the study of biofilm pathogenesis. The demonstration of liposomal amphotericin B lock therapy as an effective strategy for treating C. albicans catheter-associated infections may have significant clinical implications, and a large prospective, randomized clinical trial should be performed in the near future.