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.     

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.     

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.     

Activities of Fluconazole,Caspofungin, Anidulafungin,and Amphotericin B on Planktonic and Biofilm Candida Species Determined by Microcalorimetry

We investigated the activities of fluconazole, caspofungin, anidulafungin, and amphotericin B against Candida species in planktonic form and biofilms using a highly sensitive assay measuring growth-related heat production (microcalorimetry). C. albicans, C. glabrata, C. krusei, and C. parapsilosis were tested, and MICs were determined by the broth microdilution method. The antifungal activities were determined by isothermal microcalorimetry at 37°C in RPMI 1640. For planktonic Candida, heat flow was measured in the presence of antifungal dilutions for 24 h. Candida biofilm was formed on porous glass beads for 24 h and exposed to serial dilutions of antifungals for 24 h, and heat flow was measured for 48 h. The minimum heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration reducing the heat flow peak by ≥50% (≥90% for amphotericin B) at 24 h for planktonic Candida and at 48 h for Candida biofilms (measured also at 24 h). Fluconazole (planktonic MHICs, 0.25 to >512 μg/ml) and amphotericin B (planktonic MHICs, 0.25 to 1 μg/ml) showed higher MHICs than anidulafungin (planktonic MHICs, 0.015 to 0.5 μg/ml) and caspofungin (planktonic MHICs, 0.125 to 0.5 μg/ml). Against Candida species in biofilms, fluconazole''s activity was reduced by >1,000-fold compared to its activity against the planktonic counterparts, whereas echinocandins and amphotericin B mainly preserved their activities. Fluconazole induced growth of planktonic C. krusei at sub-MICs. At high concentrations of caspofungin (>4 μg/ml), paradoxical growth of planktonic C. albicans and C. glabrata was observed. Microcalorimetry enabled real-time evaluation of antifungal activities against planktonic and biofilm Candida organisms. It can be used in the future to evaluate new antifungals and antifungal combinations and to study resistant strains.     

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.     

Caspofungin at Catheter Lock Concentrations Eradicates Mature Biofilms of Candida lusitaniae and Candida guilliermondii

The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).     

Correction: Characterization of an exopolysaccharide from probiont Enterobacter faecalis MSI12 and its effect on the disruption of Candida albicans biofilm

Correction for ‘Characterization of an exopolysaccharide from probiont Enterobacter faecalis MSI12 and its effect on the disruption of Candida albicans biofilm’ by G. Seghal Kiran et al., RSC Adv., 2015, 5, 71573–71585, DOI: 10.1039/C5RA10302A.

The authors regret that incorrect versions of Fig. 7 and ​and88 were included in the original article. The correct versions of Fig. 7 and ​and88 are presented below with updated captions.Open in a separate windowFig. 7Phase contrast micrographs showing biofilm disruption potential of MSI12-EPS on C. albicans. The C. albicans biofilm was developed on a cover glass and then it was treated with varying concentrations of EPS and fluconazole ranging from 50–250 μg. The treated cover glass was stained with crystal violet and observed under a phase-contrast microscope (Nikon) at ×40 magnification. A – Control biofilm, B1 – 50 mg fluconazole, B2 – 50 μg MSI12-EPS, C1 – 100 mg fluconazole, C2 – 100 μg MSI12-EPS, D1 – 150 mg fluconazole, D2 – 150 μg MSI12-EPS, E1 – 200 mg fluconazole, E2 – 200 μg MSI12-EPS and F1 – 250 mg fluconazole, F2 – 250 μg MSI12-EPS. The images were recorded using a uniform scale of 10 μm which is shown in the image panels.Open in a separate windowFig. 8Confocal laser scanning micrographs showing biofilm disruption potential of MSI12-EPS on C. albicans. The pre-formed biofilm was treated for 24 h with EPS and fluconazole of varying concentrations ranging from 50–250 μg. Untreated biofilms were used as controls and the biofilm coverage thus formed on glass slides were stained with 0.1% acridine orange and subjected to visualization in a CLSM (LSM 710, Carl Zeiss). A – Control biofilm, B1 – 50 mg fluconazole, B2 – 50 μg MSI12-EPS, C1 – 100 mg fluconazole, C2 – 100 μg MSI12-EPS, D1 – 150 mg fluconazole, D2 – 150 μg MSI12-EPS, E1 – 200 mg fluconazole, E2 – 200 μg MSI12-EPS and F1 – 250 mg fluconazole, F2 – 250 μg MSI12-EPS.Accordingly, the experimental methods followed in the phase contrast microscopy and large-size images of Fig. 7 have been provided in the ESI. The ESI has been updated online to reflect this change.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Derivatives of the Mouse Cathelicidin-Related Antimicrobial Peptide (CRAMP) Inhibit Fungal and Bacterial Biofilm Formation

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 μM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 μM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.     

Effect of Growth Rate on Resistance of Candida albicans Biofilms to Antifungal Agents

A perfused biofilm fermentor, which allows growth-rate control of adherent microbial populations, was used to assess whether the susceptibility of Candida albicans biofilms to antifungal agents is dependent on growth rate. Biofilms were generated under conditions of glucose limitation and were perfused with drugs at a high concentration (20 times the MIC). Amphotericin B produced a greater reduction in the number of daughter cells in biofilm eluates than ketoconazole, fluconazole, or flucytosine. Similar decreases in daughter cell counts were observed when biofilms growing at three different rates were perfused with amphotericin B. In a separate series of experiments, intact biofilms, resuspended biofilm cells, and newly formed daughter cells were removed from the fermentor and were exposed to a lower concentration of amphotericin B for 1 h. The susceptibility profiles over a range of growth rates were then compared with those obtained for planktonic cells grown at the same rates under glucose limitation in a chemostat. Intact biofilms were resistant to amphotericin B at all growth rates tested, whereas planktonic cells were resistant only at low growth rates (≤0.13 h⁻¹). Cells resuspended from biofilms were less resistant than intact biofilm populations but more resistant than daughter cells; the susceptibilities of both these cell types were largely independent of growth rate. Our findings indicate that the amphotericin B resistance of C. albicans biofilms is not simply due to a low growth rate but depends on some other feature of the biofilm mode of growth.     

In Vitro Activities of Antibiotics and Antimicrobial Cationic Peptides Alone and in Combination against Methicillin-Resistant Staphylococcus aureus Biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) strains are most often found as hospital- and community-acquired infections. The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of bacteria that form strong biofilms. We investigated the in vitro activities of antibiotics (daptomycin, linezolid, teichoplanine, azithromycin, and ciprofloxacin) and antimicrobial cationic peptides {AMPs; indolicidin, CAMA [cecropin (1-7)–melittin A (2-9) amide], and nisin} alone or in combination against MRSA ATCC 43300 biofilms. The MICs and minimum biofilm eradication concentrations (MBECs) were determined by the broth microdilution technique. Antibiotic and AMP combinations were assessed using the checkerboard technique. For MRSA planktonic cells, MICs of antibiotics and AMPs ranged between 0.125 and 512 and 8 and 16 mg/liter, respectively, and the MBEC values were between 512 and 5,120 and 640 mg/liter, respectively. With a fractional inhibitory concentration of ≤0.5 as the borderline, synergistic interactions against MRSA biofilms were frequent with almost all antibiotic-antibiotic and antibiotic-AMP combinations. Against planktonic cells, they generally had an additive effect. No antagonism was observed. All of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 MIC and biofilm formation at 1× MIC. Biofilm-associated MRSA was not affected by therapeutically achievable concentrations of antimicrobial agents. Use of a combination of antimicrobial agents can provide a synergistic effect, which rapidly enhances antibiofilm activity and may help prevent or delay the emergence of resistance. AMPs seem to be good candidates for further investigations in the treatment of MRSA biofilms, alone or in combination with antibiotics.     

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl₃, or FeCl₃ alone. A preformed biofilm was exposed to DFP with or without FeCl₃. The DFP and DFS MIC₅₀ against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl₃ was lower than that in the controls (P < 0.05 to 0.001). FeCl₃ at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl₃ at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl₃ alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl₃ only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.     

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.     

Mechanism of Fluconazole Resistance in Candida krusei

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of C. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.     

Activity of Voriconazole, a New Triazole, Combined with Neutrophils or Monocytes against Candida albicans: Effect of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

The antifungal activity of voriconazole (VCZ) was tested against Candida albicans in the absence or presence of polymorphonuclear neutrophils (PMN) or monocytes. In some experiments, VCZ was compared to fluconazole (FCZ). On a weight basis, VCZ was 10-fold more efficacious than FCZ against C. albicans Sh27. Against an FCZ-resistant isolate, VCZ at 1 μg/ml produced the same fungistasis as FCZ at 20 μg/ml. VCZ at 0.1 μg/ml collaborated with PMN for enhanced killing to the same extent as FCZ at 1.0 μg/ml. Granulocyte-colony-stimulating factor (G-CSF) enhanced the candidacidal activity of PMN, and it increased the collaboration of PMN with VCZ for killing. Granulocyte-macrophage (GM)-CSF also significantly enhanced both the killing by PMN and the collaboration of PMN with VCZ for killing. VCZ collaborated with monocytes for enhanced killing of C. albicans Sh27, and GM-CSF increased this collaboration. Taken together, these data show that VCZ is more potent than FCZ against C. albicans isolates, alone and in collaboration with PMN or monocytes for enhanced killing. In addition, G-CSF- or GM-CSF-activated PMN and monocytes have enhanced collaboration with VCZ compared to that of unstimulated phagocytes with VCZ.     

Effects of Azithromycin in Combination with Vancomycin,Daptomycin, Fosfomycin,Tigecycline, and Ceftriaxone on Staphylococcus epidermidis Biofilms

Staphylococcal biofilms on surgical implants are the underlying cause of a lack of response to antimicrobial treatment. We investigated the effects of vancomycin (VAN), daptomycin (DAP), fosfomycin (FOS), tigecycline (TGC), and ceftriaxone (CRX), alone and in combination with azithromycin (AZI), on established biofilms of Staphylococcus epidermidis. Biofilms were studied using the static microtiter plate model with established S. epidermidis biofilms, with an initial inoculum of 10⁶/ml in 96-well polystyrene flat-bottom microtiter plates. Biofilms were inoculated with VAN, DAP, FOS, TGC, or CRX at two concentrations, alone or in combination with AZI (2, 512, or 1,024 mg/liter). To assess the reduction in biomass, the optical density ratio (ODr), calculated as (optical density [OD] of the treated biofilm)/(OD of the untreated biofilm, taken as 1), was used. For antibacterial efficacy, the viable bacterial count was used. Reductions in the biofilm ODr were observed for VAN (15 and 40 mg/liter) and FOS (200 mg/liter) only (ODr [mean ± standard deviation] for VAN at 15 and 40 mg/liter, 0.77 ± 0.32 and 0.8 ± 0.35, respectively; ODr for FOS at 200 mg/liter, 0.78 ± 0.26; P < 0.05), but not for DAP (2 and 5 mg/liter), TGC (0.2 and 2 mg/liter), or CRX (600 and 2,400 mg/liter). The addition of AZI had no further effect on the ODr, but a significant reduction of bacterial growth was achieved with high doses of AZI plus TGC or AZI plus CRX (a 3-log count reduction for AZI at 1,024 mg/liter plus CRX at 600 mg/liter and for AZI at 512 or 1,024 mg/liter plus CRX at 2,400 mg/liter; a 2-log count reduction for AZI at 512 or 1,024 mg/liter plus TGC at 2 mg/liter [P < 0.05]). No significant reduction in bacterial growth was observed for FOS (50 and 200 mg/liter), DAP (2 and 5 mg/liter), or TGC (0.2 mg/liter) in combination with AZI. None of the antibiotics at either concentration reduced the bacterial count of the biofilms when used alone. Thus, the use of a combination of AZI plus TGC, FOS, or CRX at high concentrations has little effect on biofilm density but significantly reduces bacterial growth.Staphylococcus epidermidis is the leading pathogen causing infections of surgical implants. Given the high incidence of fracture fixation devices, 2 million per annum, the number of implant infections amounts to 100,000 per year in the United States (10). Many of these infections are associated with biofilms formed by staphylococci on implant surfaces (9, 26, 34). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. The two consequences of biofilm formation on implant surfaces are increased resistance to antimicrobial agents and frequent failure of conventional antimicrobial therapy. This resistance of bacteria within biofilms is attributed to a possible barrier function of the biofilm, binding of the antimicrobial agents within the matrix, and the metabolic change in the bacterial cells. Thus, infection of medical implants is associated with considerable morbidity and costs due to loss of mobility, nonproductive time, and health care (10, 18, 35).The antimicrobial agents most widely used for staphylococcal infections are beta-lactam antibiotics, primarily cephalosporins, and in the case of beta-lactam resistance, vancomycin (23, 28). Alternative agents are intravenous fosfomycin, a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, and two newer antibiotics, the glycylcycline tigecycline and the cyclic lipopeptide daptomycin (14). Daptomycin, which is highly active against gram-positive cocci resistant to commonly used antibiotics, including methicillin (meticillin)-resistant staphylococci, causes membrane changes in the bacteria, whereas tigecycline inhibits protein synthesis (20). Although macrolides are not commonly used in the treatment of staphylococcal infections, clinical and experimental data suggest that azithromycin decreases biofilm formation and enhances the efficacy of other antibiotics for patients with cystic fibrosis and Pseudomonas infections (17, 19).The MICs of antimicrobial agents tested on bacterial biofilms are dramatically increased, up to concentrations >1,000 times the MICs for staphylococci under planktonic conditions (5). However, there is some evidence that higher antibiotic concentrations reduce biofilms and bacterial growth, whereas standard concentrations are not effective (24, 30). In a previous study, we demonstrated that antibiotic concentrations equivalent to 100 times the MIC under planktonic conditions did not decrease established staphylococcal biofilms (16). To investigate if even higher concentrations will overcome the bacterial resistance within biofilms and reduce biofilm thickness and bacterial growth, static biofilms of clinical S. epidermidis isolates causing implant infections and catheter-associated bacteremia were incubated with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone at two concentrations. To explore the additional effect of the macrolide azithromycin (22) on S. epidermidis biofilms, we treated the biofilms with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone in combination with azithromycin at three concentrations (2, 512, and 1,024 mg/liter).(Part of this research was presented as a poster at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [29a].)     

Structure-In Vitro Activity Relationships of Pentamidine Analogues and Dication-Substituted Bis-Benzimidazoles as New Antifungal Agents

Twenty analogues of pentamidine, 7 primary metabolites of pentamidine, and 30 dicationic substituted bis-benzimidazoles were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. A majority of the compounds had MICs at which 80% of the strains were inhibited (MIC₈₀s) comparable to those of amphotericin B and fluconazole. Unlike fluconazole, many of these compounds were found to have potent fungicidal activity. The most potent compound against C. albicans had an MIC₈₀ of ≤0.09 μg/ml, and the most potent compound against C. neoformans had an MIC₈₀ of 0.19 μg/ml. Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. It is clear from the data presented here that further studies on the structure-activity relationships, mechanisms of action and toxicities, and in vivo efficacies of these compounds are warranted to determine their clinical potential.     

Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [K_d] of 10 and 13 nM), whereas fluconazole bound weakly (K_d of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC₅₀] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC₅₀, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.