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.     

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.     

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.     

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.     

Flow Cytometry-Based Method To Detect Persisters in Candida albicans

Candida albicans biofilms contain a subpopulation whose members are defined as persisters, displaying great tolerance of fungicides. To directly observe such persisters, an effective method using green fluorescent protein (GFP) strain labeling by mutation of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (TDH3), combined with propidium iodide (PI) staining, was established. Amphotericin B-tolerant persisters harbor the characteristics of both GFP positivity [GFP (+)] and propidium iodide (PI) negativity [PI (−)], which are easily visualized using a fluorescence microscope and measured by flow cytometry.     

Cell density and cell aging as factors modulating antifungal resistance of Candida albicans biofilms

Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 10⁸ cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.     

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.     

Biofilm-formation capability depends on environmental oxygen concentrations in Candida species

BackgroundAlthough oxygen concentrations inside of the human body vary depending on organs or tissues, few reports describe the relationships between biofilm formation of Candida species and oxygen concentrations. In this study, we investigated the biofilm-forming capabilities of Candida species under various oxygen conditions.MethodsWe evaluated the adhesion and biofilm formation of Candida albicans and C. tropicalis under aerobic, microaerobic (oxygen concentration 5%), or anaerobic conditions. We also examined how oxygen concentration affects adhesion/maturation by changing adhesion/maturation phase conditions. We used crystal violet assay to estimate the approximate biofilm size, performed microscopic observation of biofilm morphology, and evaluated adhesion-associated gene expression.ResultsThe adhered amount was relatively small except for a clinical strain of C. tropicalis. Our biofilm-formation analysis showed that C. albicans formed a higher-size biofilm under aerobic conditions, while C. tropicalis favored microaerobic conditions to form mature biofilms. Our microscopic observations were consistent with these biofilm-formation analysis results. In particular, C. tropicalis exhibited more hyphal formation under microaerobic conditions. By changing the adhesion/maturation phase conditions, we represented that C. albicans had favorable biofilm-formation capability under aerobic conditions, while C. tropicalis showed enhanced biofilm formation under microaerobic adhesion conditions. In good agreement with these results, the C. tropicalis adhesion-associated gene expression tended to be higher under microaerobic or anaerobic conditions.ConclusionsC. albicans favored aerobic conditions to form biofilms, whereas C. tropicalis showed higher biofilm-formation ability and promoted hyphal growth under microaerobic conditions. These results indicate that favorable oxygen conditions significantly differ for each Candida species.     

Patients with Long-Term Oral Carriage Harbor High-Persister Mutants of Candida albicans

Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.Candida species are opportunistic pathogens that are typically present in the oral cavities of healthy individuals (2, 14, 24). In immunocompromised patients, the severity of Candida infection can range from a superficial annoyance to a life-threatening systemic infection of the organs and sepsis. While superficial infections of the oral or vaginal mucosa are easily treatable with azoles, 5 to 8% resist therapy, producing relapses (11, 30). Systemic invasive fungal infections, characterized by the hyphal growth of Candida albicans, are the cause of high rates of morbidity and mortality, which approach 40% (8). Difficult-to-treat Candida infections also occur on prosthetic devices, such as catheters and heart valves, and an infected prosthesis requires device removal to avoid systemic infection (22).Candida forms biofilms on the surfaces of mucosal tissues and prostheses that are highly tolerant to antifungal agents (18, 26). The recalcitrance of biofilm infections to antimicrobials is not obvious, since planktonic populations of disease-causing strains can be highly susceptible to antifungals, including azoles, echinocandins, and amphotericin B (AMB) (16).We reported that upon attachment, C. albicans forms a small subpopulation (∼1%) of persister cells that are completely tolerant to the currently used systemic antifungals (16) and resemble well-characterized dormant persisters formed by pathogenic bacteria (12, 13, 18, 27, 28, 31, 32). The concentration-dependent killing of a C. albicans biofilm with a fungicidal agent such as AMB shows a sharply biphasic pattern, with the bulk of the cells rapidly dying and a small plateau of surviving persisters being present. Vital staining shows live persister cells present in a biofilm killed by exposure to a high level of AMB, and these cells can be sorted out from the bulk. Surviving persisters produce a new biofilm with a similarly small population of persisters, indicating that these cells are not classical resistant mutants but phenotypic variants of the wild type. Persisters exhibit multidrug tolerance, which is a hallmark of a biofilm infection.Attachment to a surface is an important step in fungal pathogenesis, including pathogenesis resulting in vaginitis, oral thrush, and catheter biofilm infections (6, 10, 15). It seems that persisters, which form upon attachment of the pathogen to a surface, may play an important role in the tolerance of Candida infections to antifungals.In Escherichia coli, the periodic application of a high concentration of a bactericidal antibiotic in vitro leads to the selection of high-persister (hip) mutants (20, 21). Importantly, these mutants have the same MIC as the wild type, but they produce considerably more persister cells. While the persister phenotype itself is not due to a mutation, high-persister strains carry mutations that cause an increased incidence of persisters. One of these mutants was mapped to an allele of a hipA gene coding for a toxin of the hipBA toxin/antitoxin module (13, 21). The mechanistic basis of HipA-dependent persister formation was recently identified (27). HipA is a protein kinase (3) that phosphorylates elongation factor EF-Tu, which leads to the inhibition of protein synthesis. This creates a dormant, persister state. The HipA7 allele, which causes the high persistence of the hip mutant, apparently has decreased binding to the antitoxin HipB, which leads to increased persister production.We reasoned that a similar selection for high-persister mutants is likely to occur in vivo, especially in cases of recalcitrant infections, where pathogens are periodically exposed to high levels of antimicrobial compounds. With this in mind, we tested a collection of 150 clinical isolates of Candida species for their persister levels. The strains were obtained from cancer patients who received daily topical chlorhexidine (CHX) treatment. We report that the strains isolated from patients with long-term Candida carriage had increased levels of surviving persisters.     

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.     

Candida albicans biofilms produce antifungal-tolerant persister cells

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.     

Sustained Nitric Oxide-Releasing Nanoparticles Induce Cell Death in Candida albicans Yeast and Hyphal Cells,Preventing Biofilm Formation In Vitro and in a Rodent Central Venous Catheter Model

Candida albicans is a leading nosocomial pathogen. Today, candidal biofilms are a significant cause of catheter infections, and such infections are becoming increasingly responsible for the failure of medical-implanted devices. C. albicans forms biofilms in which fungal cells are encased in an autoproduced extracellular polysaccharide matrix. Consequently, the enclosed fungi are protected from antimicrobial agents and host cells, providing a unique niche conducive to robust microbial growth and a harbor for recurring infections. Here we demonstrate that a recently developed platform comprised of nanoparticles that release therapeutic levels of nitric oxide (NO-np) inhibits candidal biofilm formation, destroys the extracellular polysaccharide matrices of mature fungal biofilms, and hinders biofilm development on surface biomaterials such as the lumen of catheters. We found NO-np to decrease both the metabolic activity of biofilms and the cell viability of C. albicans in vitro and in vivo. Furthermore, flow cytometric analysis found NO-np to induce apoptosis in biofilm yeast cells in vitro. Moreover, NO-np behave synergistically when used in combination with established antifungal drug therapies. Here we propose NO-np as a novel treatment modality, especially in combination with standard antifungals, for the prevention and/or remediation of fungal biofilms on central venous catheters and other medical devices.     

Biofilm Production and Antibiofilm Activity of Echinocandins and Liposomal Amphotericin B in Echinocandin-Resistant Yeast Species

The echinocandins and liposomal amphotericin B are active against biofilm produced by echinocandin-susceptible Candida strains. However, few data have been reported on the production of biofilm by echinocandin-resistant isolates and their antifungal susceptibility. We studied the production of biofilm by fks mutant Candida strains and intrinsically echinocandin-resistant non-Candida isolates and the susceptibility of both entities to liposomal amphotericin B and echinocandins. We analyzed the production of biofilm by isolates from patients with fungemia (fks mutant Candida, n = 5; intrinsically echinocandin-resistant non-Candida, n = 12; and Candida wild type, n = 10). Biofilm formation was measured to classify strains according to biomass (crystal violet assay) and metabolic activity (XTT reduction assay). Preformed biofilms were tested against liposomal amphotericin B, caspofungin, micafungin, and anidulafungin. The sessile MIC was defined as the antifungal concentration yielding a 50% or 80% reduction in the metabolic activity of the biofilm compared to that of the growth control (SMIC₅₀ and SMIC₈₀, respectively). fks mutant Candida isolates formed biofilms in a fashion similar to that of Candida wild-type strains. The echinocandins had the highest activity against biofilms formed by wild-type Candida isolates, followed by fks mutant Candida isolates and non-Candida isolates. Liposomal amphotericin B had the highest activity against fks mutant Candida biofilms. The formation of biofilm by echinocandin-resistant strains was similar to that of wild-type strains, although resistance to echinocandins remained high.     

Micafungin at Physiological Serum Concentrations Shows Antifungal Activity against Candida albicans and Candida parapsilosis Biofilms

We assessed the in vitro activity of micafungin against preformed Candida biofilms by measuring the concentration of drug causing the most fungal damage and inhibition of regrowth. We studied 37 biofilm-producing Candida spp. strains from blood cultures. We showed that micafungin was active against planktonic and sessile forms of Candida albicans strains and moderately active against Candida parapsilosis sessile cells. Concentrations of micafungin above 2 μg/ml were sufficiently high to inactivate regrowth of Candida sessile cells.     

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.     

Iron-Limited Biofilms of Candida albicans and Their Susceptibility to Amphotericin B

Biofilms of Candida albicans were grown in vitro under iron limitation and at a low growth rate to simulate conditions for implant-associated biofilms in vivo. Their properties were compared with those of glucose-limited biofilms grown under analogous conditions. At steady state, the adherent cell populations of iron-limited biofilms were double those of glucose-limited biofilms, although the growth rates were similar (0.038 to 0.043 h⁻¹). Both biofilm types were resistant to amphotericin B, but daughter cells from iron-limited biofilms were significantly more susceptible to the drug than those from glucose-limited biofilms.     

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms

Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC₅₀s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC₅₀ from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.     

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.