In Vitro Analysis of Finasteride Activity against Candida albicans Urinary Biofilm Formation and Filamentation

Candida albicans is the 3rd most common cause of catheter-associated urinary tract infections, with a strong propensity to form drug-resistant catheter-related biofilms. Due to the limited efficacy of available antifungals against biofilms, drug repurposing has been investigated in order to identify novel agents with activities against fungal biofilms. Finasteride is a 5-α-reductase inhibitor commonly used for the treatment of benign prostatic hyperplasia, with activity against human type II and III isoenzymes. We analyzed the Candida Genome Database and identified a C. albicans homolog of type III 5-α-reductase, Dfg10p, which shares 27% sequence identity and 41% similarity to the human type III 5-α-reductase. Thus, we investigated finasteride for activity against C. albicans urinary biofilms, alone and in combination with amphotericin B or fluconazole. Finasteride alone was highly effective in the prevention of C. albicans biofilm formation at doses of ≥16 mg/liter and the treatment of preformed biofilms at doses of ≥128 mg/liter. In biofilm checkerboard analyses, finasteride exhibited synergistic activity in the prevention of biofilm formation in a combination of 4 mg/liter finasteride with 2 mg/liter fluconazole. Finasteride inhibited filamentation, thus suggesting a potential mechanism of action. These results indicate that finasteride alone is highly active in the prevention of C. albicans urinary biofilms in vitro and has synergistic activity in combination with fluconazole. Further investigation of the clinical utility of finasteride in the prevention of urinary candidiasis is warranted.     

Candida albicans and Staphylococcus aureus Form Polymicrobial Biofilms: Effects on Antimicrobial Resistance

Candida albicans readily forms biofilms on the surface on indwelling medical devices, and these biofilms serve as a source of local and systemic infections. It is estimated that 27% of nosocomial C. albicans bloodstream infections are polymicrobial, with Staphylococcus aureus as the third most common organism isolated in conjunction with C. albicans. We tested whether S. aureus and C. albicans are able to form a polymicrobial biofilm. Although S. aureus formed poor monoculture biofilms in serum, it formed a substantial polymicrobial biofilm in the presence of C. albicans. In terms of architecture, S. aureus formed microcolonies on the surface of the biofilm, with C. albicans serving as the underlying scaffolding. In addition, S. aureus matrix staining revealed a different phenotype in polymicrobial versus monomicrobial biofilms, suggesting that S. aureus may become coated in the matrix secreted by C. albicans. S. aureus resistance to vancomycin was enhanced within the polymicrobial biofilm, required viable C. albicans, and was in part mediated by C. albicans matrix. However, the growth or sensitivity to amphotericin B of C. albicans is not altered in the polymicrobial biofilm.There is increasing evidence in the literature for the importance of polymicrobial infections in which microorganisms interact in a synergistic or inhibitory fashion, impacting pathogenesis and the health of the patient. It was originally estimated that over half of infections originated from biofilms (12). However, the NIH currently estimates that biofilms account for over 80% of all infections in the body (NIH SBIR/STTR Study and Control of Microbial Biofilms program announcement, release date 21 April 1999, http://grants.nih.gov/grants/guide/pa-files/PA-99-084.html). Biofilms are communities of microbes embedded in a polysaccharide matrix adhered to a biotic or abiotic surface. The biofilm-associated microorganisms are refractory to both antimicrobial agents and the host immune response. Polymicrobial biofilms represent an understudied and clinically relevant health problem, with the potential to serve as an infectious reservoir for a variety of microorganisms, including bacteria and fungi.Biofilms can form on indwelling medical devices and serve as a source of nosocomial bloodstream infections, which prolong hospitalization and are the 10th leading cause of death in the United States (30). Candida albicans is the fourth leading cause of bloodstream infections and the third most commonly isolated organism from intravascular catheters and is associated with the highest incidence of mortality (13, 30). C. albicans readily forms biofilms on a wide variety of polymers used to make indwelling medical devices, such as dental materials, stents, shunts, prostheses (voice, heart valve, knee, etc.), implants (lens, breast, denture, penile, etc.), endotracheal tubes, pacemakers, and catheters (reviewed in reference 23). There is some evidence to suggest that a large proportion of device-related Candida albicans infections involve biofilms (14, 15, 23). In a prospective study of catheter colonization, C. albicans ranked second in the ratio of colonization to invasive disease (13). C. albicans biofilms have a unique gene expression pattern (18, 24, 31) and are more resistant to antifungal treatment than planktonic cells (23, 26). A unique feature of C. albicans biofilms is the morphological heterogeneity of the biofilm cells, which results in a complex three-dimensional biofilm architecture (11). C. albicans biofilm formation is initiated upon contact with an appropriate polymeric surface under morphogenesis-inducing growth conditions. Serum is the classical clinically relevant inducer of morphogenesis, although other media can be used in vitro. During early biofilm formation, yeast cells adhere to an appropriate surface and initiate germ tube formation. The intermediate phase is characterized by continued hyphal elongation and extracellular matrix production, which is composed primarily of glucose along with proteins and other sugars (2). Mature biofilms consist of a yeast base, with hyphal elements encased in matrix extending away from the surface forming a sticky net-like structure (11). Newly formed daughter yeast cells grow out of hyphal elements and are released, seeding new niches for biofilm formation or infection.It is estimated that 27% of nosocomial C. albicans bloodstream infections are polymicrobial, with Staphylococcus aureus as the third most common organism isolated in conjunction with C. albicans (22). Interestingly, the combined effect of C. albicans and S. aureus results in synergism and increased mortality in mice (5-9). Although the infectious parameters of polymicrobial infections in humans are not well characterized, eliminating the infection may involve a more complex antimicrobial regimen. It has previously been demonstrated that a mixed species biofilm of C. albicans and Staphylococcus epidermidis enhances the growth of S. epidermidis and increases the resistance of S. epidermidis to vancomycin (1, 16). It is worth mentioning that biofilm formation in S. epidermidis and biofilm formation in S. aureus are not equivalent. Compared with S. epidermidis, S. aureus does not form biofilms as readily on abiotic surfaces, requiring precoating and nutrient supplementation (10). However, S. aureus is a more clinically important pathogen, with higher rates of device-related systemic infection and mortality (reviewed in references 20 and 25). Therefore, we sought to determine whether C. albicans and S. aureus could form polymicrobial biofilms and whether these biofilms exhibited altered antimicrobial sensitivity.     

Antibiofilm Activity of Electrical Current in a Catheter Model

Catheter-associated infections are difficult to treat with available antimicrobial agents because of their biofilm etiology. We examined the effect of low-amperage direct electrical current (DC) exposure on established bacterial and fungal biofilms in a novel experimental in vitro catheter model. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida parapsilosis biofilms were grown on the inside surfaces of polyvinyl chloride (PVC) catheters, after which 0, 100, 200, or 500 μA of DC was delivered via intraluminally placed platinum electrodes. Catheter biofilms and intraluminal fluid were quantitatively cultured after 24 h and 4 days of DC exposure. Time- and dose-dependent biofilm killing was observed with all amperages and durations of DC administration. Twenty-four hours of 500 μA of DC sterilized the intraluminal fluid for all bacterial species studied; no viable bacteria were detected after treatment of S. epidermidis and S. aureus biofilms with 500 μA of DC for 4 days.     

Glyceryl Trinitrate Complements Citrate and Ethanol in a Novel Antimicrobial Catheter Lock Solution To Eradicate Biofilm Organisms

Antimicrobial catheter lock therapy is practiced to prevent lumenal-sourced infections of central venous catheters. Citrate has been used clinically as an anticoagulant in heparin-free catheter locks. Ethanol has also been widely studied as an antimicrobial lock solution component. This study reports on the synergy of glyceryl trinitrate (GTN) with citrate and ethanol in rapidly eradicating methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans biofilms in an in vitro model for catheter biofilm colonization. GTN has a long history of intravenous use as a hypotensive agent. It is potentially attractive as a component of a catheter lock solution because its physiologic half-life is quite short and its metabolic pathways are known. A lock containing 7% citrate and 20% ethanol required 0.01% GTN to fully eradicate biofilms of all test organisms within 2 h in the model. This GTN concentration is below the levels where clinically significant hypotensive effects are expected.     

Antifungal Lock Therapy

The widespread use of intravascular devices, such as central venous and hemodialysis catheters, in the past 2 decades has paralleled the increasing incidence of catheter-related bloodstream infections (CR-BSIs). Candida albicans is the fourth leading cause of hospital-associated BSIs. The propensity of C. albicans to form biofilms on these catheters has made these infections difficult to treat due to multiple factors, including increased resistance to antifungal agents. Thus, curing CR-BSIs caused by Candida species usually requires catheter removal in addition to systemic antifungal therapy. Alternatively, antimicrobial lock therapy has received significant interest and shown promise as a strategy to treat CR-BSIs due to Candida species. The existing in vitro, animal, and patient data for treatment of Candida-related CR-BSIs are reviewed. The most promising antifungal lock therapy (AfLT) strategies include use of amphotericin, ethanol, or echinocandins. Clinical trials are needed to further define the safety and efficacy of AfLT.     

Targeting Fibronectin To Disrupt In Vivo Candida albicans Biofilms

New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation.     

Effects of lactoferricin B against keratitis-associated fungal biofilms

Biofilms are considered as the most important developmental characteristics in ocular infections. Biofilm eradication is a major challenge today to overcome the incidence of drug resistance. This report demonstrates the in vitro ability of biofilm formation on contact lens by three common keratitis-associated fungal pathogens, namely, Aspergillus fumigatus, Fusarium solani, and Candida albicans. Antifungal sensitivity testing performed for both planktonic cells and biofilm revealed the sessile phenotype to be resistant at MIC levels for the planktonic cells and also at higher concentrations. A prototype lens care solution was also found to be partially effective in eradication of the mature biofilm from contact lenses. Lactoferricin B (Lacf, 64 μg/ml), an antimicrobial peptide, exhibited almost no effect on the sessile phenotype. However, the combinatory effect of Lacf with antifungals against planktonic cells and biofilms of three fungal strains that were isolated from keratitis patients exhibited a reduction of antifungal dose more than eightfold. Furthermore, the effect of Lacf in lens care solution against biofilms in which those strains formed was eradicated successfully. These results suggest that lactoferricin B could be a promising candidate for clinical use in improving biofilm susceptibility to antifungals and also as an antibiofilm-antifungal additive in lens care solution.     

High-Throughput Screening of a Collection of Known Pharmacologically Active Small Compounds for Identification of Candida albicans Biofilm Inhibitors

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.     

Reduced Vancomycin Susceptibility in an In Vitro Catheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistant S. aureus (MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilms in vitro and nonsusceptibility to VAN in endovascular infection in vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of such in vivo VAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in an in vitro catheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in the in vitro catheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.     

Efficacy of liposomal amphotericin B against four species of Candida biofilms in an experimental mouse model of intravascular catheter infection

The formation of Candida biofilms on implanted medical devices is crucial to the development of infections and an important clinical problem because of elevated resistance to antifungals. The aim of this study was to compare the in vitro activity of liposomal amphotericin B (L-AMB) and micafungin (MCFG) against four species of Candida biofilms, and the efficacy of systemic plus lock therapy with L-AMB and MCFG in a Candida biofilm-associated catheter infection model. An XTT-reduction assay was used to measure the metabolic activity of the biofilms to evaluation of in vitro antibiofilm activity. MCFG had better in vitro activity than L-AMB against Candida glabrata biofilms, whereas L-AMB had better activity than MCFG against Candida albicans and Candida tropicalis biofilms. L-AMB and MCFG had comparable efficacy against Candida parapsilosis biofilms. In an in vitro lock therapy model, 2 mg/ml L-AMB, unlike 2 mg/ml MCFG, significantly reduced the metabolic activity of all the strains of biofilms by >96%. Systemic and intraluminal lock treatment with L-AMB for 3-days resulted in more than about 2 log₁₀ reduction of Candida compared with that of systemic treatment and the control group in the C. albicans SP-20012, C. glabrata SP-20040, C. glabrata SP-20131, C. parapsilosis SP-20137, and C. tropicalis SP-20047 infection models. L-AMB was more effective at eradicating Candida biofilms in 3-day course of systemic and lock therapy than MCFG. L-AMB may be useful for the treatment of catheter-related Candida biofilm infections, but this finding will need to be confirmed by further studies including a long treatment duration.     

Species-Specific and Drug-Specific Differences in Susceptibility of Candida Biofilms to Echinocandins: Characterization of Less Common Bloodstream Isolates

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.     

Fungal β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia coli in a Polymicrobial E. coli/Candida albicans Biofilm

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.     

Efficacy of Ethanol against Candida albicans and Staphylococcus aureus Polymicrobial Biofilms

Candida albicans, an opportunistic fungus, and Staphylococcus aureus, a bacterial pathogen, are two clinically relevant biofilm-forming microbes responsible for a majority of catheter-related infections, with such infections often resulting in catheter loss and removal. Not only do these pathogens cause a substantial number of nosocomial infections independently, but also they are frequently found coexisting as polymicrobial biofilms on host and environmental surfaces. Antimicrobial lock therapy is a current strategy to sterilize infected catheters. However, the robustness of this technique against polymicrobial biofilms has remained largely untested. Due to its antimicrobial activity, safety, stability, and affordability, we tested the hypothesis that ethanol (EtOH) could serve as a potentially efficacious catheter lock solution against C. albicans and S. aureus biofilms. Therefore, we optimized the dose and time necessary to achieve killing of both monomicrobial and polymicrobial biofilms formed on polystyrene and silicone surfaces in a static microplate lock therapy model. Treatment with 30% EtOH for a minimum of 4 h was inhibitory for monomicrobial and polymicrobial biofilms, as evidenced by XTT {sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide inner salt} metabolic activity assays and confocal microscopy. Experiments to determine the regrowth of microorganisms on silicone after EtOH treatment were also performed. Importantly, incubation with 30% EtOH for 4 h was sufficient to kill and inhibit the growth of C. albicans, while 50% EtOH was needed to completely inhibit the regrowth of S. aureus. In summary, we have systematically defined the dose and duration of EtOH treatment that are effective against and prevent regrowth of C. albicans and S. aureus monomicrobial and polymicrobial biofilms in an in vitro lock therapy model.     

Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.     

Bacteriophage Cocktail for the Prevention of Biofilm Formation by Pseudomonas aeruginosa on Catheters in an In Vitro Model System

Microorganisms develop biofilms on indwelling medical devices and are associated with device-related infections, resulting in substantial morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated catheters with Pseudomonas aeruginosa bacteriophages on biofilm formation by P. aeruginosa in an in vitro model. Hydrogel-coated catheters were exposed to a 10 log₁₀ PFU ml⁻¹ lysate of P. aeruginosa phage M4 for 2 h at 37°C prior to bacterial inoculation. The mean viable biofilm count on untreated catheters was 6.87 log₁₀ CFU cm⁻² after 24 h. The pretreatment of catheters with phage reduced this value to 4.03 log₁₀ CFU cm⁻² (P < 0.001). Phage treatment immediately following bacterial inoculation also reduced biofilm viable counts (4.37 log₁₀ CFU cm⁻² reduction; P < 0.001). The regrowth of biofilms on phage-treated catheters occurred between 24 and 48 h, but supplemental treatment with phage at 24 h significantly reduced biofilm regrowth (P < 0.001). Biofilm isolates resistant to phage M4 were recovered from catheters pretreated with phage. The phage susceptibility profiles of these isolates were used to guide the development of a five-phage cocktail from a larger library of P. aeruginosa phages. The pretreatment of catheters with this cocktail reduced the 48-h mean biofilm cell density by 99.9% (from 7.13 to 4.13 log₁₀ CFU cm⁻²; P < 0.001), but fewer biofilm isolates were resistant to these phages. These results suggest the potential of applying phages, especially phage cocktails, to the surfaces of indwelling medical devices for mitigating biofilm formation by clinically relevant bacteria.Indwelling medical devices of various kinds may become colonized with microorganisms, resulting in the formation of microbial biofilms (16). Biofilm-associated organisms are tolerant to antimicrobial agents, can evade the host immune system, and can act as a nidus for infection (16). As a result, device-related infections, such as catheter-associated bloodstream infections, cause substantial morbidity and mortality among specific patient populations (9). Attributable mortality rates for healthcare-associated bloodstream infections have been estimated to be 25% (44).A number of novel strategies have been proposed to more effectively prevent and control device-associated biofilms, either by minimizing microbial attachment to device surfaces or by targeting the biofilm after it has developed. One such strategy is to use bacteriophages (phages) (17). Phages have been used for the treatment of infectious diseases in plants (26), animals (6), and humans (33, 39, 43). The use of phages to control biofilms has potential for several reasons. Phages can replicate at the site of an infection, thereby increasing in numbers where they are most required. During the lytic replication cycle, the infection of a bacterial host cell by a single phage virion will result in the production of dozens or hundreds of progeny phage, depending on the particular phage and host strains. Some phages also have been shown to produce enzymes that degrade the extracellular polymeric substance (EPS) matrix of a biofilm (23, 25). Doolittle et al. (19) showed that progeny phage will propagate radially through a biofilm. At least in theory, a single phage dose should be capable of treating a biofilm infection as progeny phage infect adjacent cells and degrade the biofilm matrix.Curtin and Donlan (13) demonstrated that a phage that is active against Staphylococcus epidermidis could be incorporated into a hydrogel coating on a catheter and significantly reduce biofilm formation by this organism in an in vitro model system. Based on those studies with S. epidermidis, we have investigated whether phages specific for Pseudomonas aeruginosa also can reduce biofilm formation by this organism in a similar in vitro model. Catheters were treated with a single phage or a combination of phages prior to, immediately following, or 24 h after inoculation with the test P. aeruginosa culture, and the effects of the phage treatments on biofilm formation and maintenance were characterized.(Portions of this paper were presented as poster no. A-011 at the 2007 American Society for Microbiology General Meeting in Toronto, Canada [20a].)     

In Vivo Efficacy of Anidulafungin against Mature Candida albicans Biofilms in a Novel Rat Model of Catheter-Associated Candidiasis

The present study demonstrates the efficacy of anidulafungin on mature Candida albicans biofilms in vivo. One hundred fifty-seven catheter fragments challenged with C. albicans were implanted subcutaneously in rats. After formation of biofilms, rats were treated with daily intraperitoneal injections of anidulafungin for 7 days. Catheters retrieved from treated animals showed reduced cell numbers compared to those retrieved from untreated and fluconazole-treated animals. Systemic administration of anidulafungin is promising for the treatment of mature C. albicans biofilms.Fungal biofilms represent a persistent source of disseminated infections in high-risk patients and are recalcitrant to antifungal therapy (11). Two classes of agents, the lipid formulations of amphotericin B and the echinocandins, appear to have a unique activity against Candida biofilms. Intraluminal lock therapy with caspofungin alone (4) or combined with systemic therapy (10) was shown to be effective against Candida biofilms in two intravascular catheter models in rabbits and mice. Anidulafungin, active against Candida biofilms in vitro (6), seems a very attractive antifungal agent to employ for a lock therapy approach since this drug was shown to induce fewer paradoxical growth effects than caspofungin and micafungin (2). Recently, we reported a novel in vivo subcutaneous Candida biofilm model in rat (8), in which biofilms develop inside catheter fragments implanted under the skin. We here report on the ability of anidulafungin to strongly reduce the number of viable cells in mature Candida biofilms in such animal model, using more than 150 infected catheters.For all experiments, the sequenced Candida albicans SC5314 strain (3) was used. Fluconazole and anidulafungin, provided by Pfizer (Groton, CT), were prepared in sterile water and dimethyl sulfoxide (DMSO), respectively. In vitro biofilm drug susceptibility assays were performed using 1-cm pieces (20 pieces per tested concentration) of serum-coated polyurethane catheters (Arrow International Reading) as previously described by Řičicová et al. (8). Biofilms were subjected to fluconazole or anidulafungin at concentrations ranging from 0.125 μg/ml to 64 μg/ml or to antifungal-free medium for 24 h. The metabolic activity of the biofilms was measured using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] reduction assay as previously described (7). Biofilm MICs were determined as the minimal drug concentration that caused ≥50% reduction in the metabolic activity of the biofilm compared to the level for the controls. In vivo biofilms were grown subcutaneously in a rat model as described by Řičicová et al. (8). Briefly, female Sprague-Dawley rats (200 g) were immunosuppressed by the addition of 1 mg/liter of dexamethasone to their drinking water. Polyurethane catheter pieces incubated overnight in serum were challenged with Candida cells (5.10⁴ cells per ml) for 90 min at 37°C and, after being washed, were implanted subcutaneously on the lower backs of the rats. Biofilms were allowed to mature for 48 h before the antifungal treatment was started. Antifungal drugs or physiological solution (control) was administrated intraperitoneally, daily, at concentrations of 125 mg/kg of body weight for fluconazole and 10 mg/kg for anidulafungin. Treatment was continued for 7 days. Eleven rats were treated with anidulafungin, 4 with fluconazole, and 7 with saline. Rats were euthanized by CO₂ inhalation prior to the removal of the catheters. Catheter fragments were washed and sonicated before biofilm quantification by CFU counting. Results were analyzed using the Mann-Whitney test (Analyze-it software).Fluconazole did not cause any reduction of metabolic activity of in vitro biofilms formed in the polyurethane catheter model even at the highest concentration of 64 μg/ml, whereas the in vitro biofilm MIC of anidulafungin was 0.25 μg/ml, with no paradoxical growth at higher concentrations. The numbers of Candida cells recovered from the implanted catheters in vivo are given in Fig. ​Fig.1.1. Despite the illustrated variation, it is noteworthy that more than 70% of catheters retrieved from treated animals contained fewer than 2 log₁₀ cells, which is below the diagnostic threshold for catheter-related infections (5). Additionally, 14 catheters (17%) retrieved from 7 out of 11 anidulafungin-treated animals were sterile. Finally, the few catheters that contained as many cells as the control biofilms were retrieved from only 2 animals out of 11, highlighting the animal-dependent variability. The mean number of CFU ± standard deviation (SD) obtained per catheter fragment of fluconazole-treated animals (3.01 ± 0.1 log₁₀ CFU/catheter fragment) was not significantly different (P = 0.94) from the mean number of Candida cells obtained from catheters of the control group (2.92 ± 0.34 log₁₀ CFU/catheter fragment). In contrast, treatment of the animals with anidulafungin significantly reduced the mean number of CFU recovered from the explanted catheter fragments (2.14 ± 0.94 log₁₀ CFU/catheter fragment) compared to the level for the control animals (P < 0.0001).Open in a separate windowFIG. 1.Effect of antifungal intraperitoneal treatment on mature Candida biofilms formed on the catheter''s lumen in a rat model. The log₁₀ numbers of CFU of Candida albicans cells cultured from each catheter in the control group, the anidulafungin group, and the fluconazole treatment group are shown. The horizontal line shows the median values for log₁₀ number of CFU obtained per catheter fragment. A significant difference was found between the anidulafungin-treated rats and the control group (*, P < 0.0001).We report here that systemic administration of anidulafungin in rats resulted in a significant reduction of C. albicans cells living within biofilms in vivo. The activity of caspofungin was previously described for two intravascular catheter animal models (4, 10). In both models, the drug was instilled intraluminally. In the subcutaneous model, anidulafungin was administrated intraperitoneally. Despite the fact that this is not the most clinically relevant mode of administration, therapeutic levels were achieved, as shown by the complete killing of the fungal population in 17% of the implanted catheters. The variability in number of CFU recovered from catheters of anidulafungin-treated animals was rather large. This might be a limitation of the model. Otherwise, this might be a reflection of the variability in clinical response that could occur while patients are treated. Intravenous treatment remains to be tested in such subcutaneous model but may lead to an even higher and more reproducible rate of killing. Our data show that the in vivo Candida albicans biofilm subcutaneous model system is very attractive for in vivo testing of the activity of antifungal drugs. In addition, the lack of in vivo activity of fluconazole on Candida biofilms reported by other groups (1, 9) was confirmed in our model. The results of this study support the use of anidulafungin for the treatment of biofilms that are not located in the intravascular compartment, but confirmation of these results in other in vivo models is certainly warranted. In conclusion, we demonstrated the activity of anidulafungin on mature Candida biofilms in an animal model. Our results are promising for the treatment of Candida biofilms on devices that cannot be readily removed from the patient.     

Inhibition of Nucleic Acid Biosynthesis Makes Little Difference to Formation of Amphotericin B-Tolerant Persisters in Candida albicans Biofilm

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml⁻¹; biofilm cells, 1 μg ml⁻¹) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml⁻¹ for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.