The activity of amphotericin B against Candida albicans is not directly associated with extracellular calcium concentration

The ability of amphotericin B to increase intracellular calcium concentrations in human cells is associated with the toxicity of this antifungal agent. The present study was performed to determine whether amphotericin B affects the influx or efflux of calcium in Candida albicans, and whether the antifungal activity of amphotericin B is dependent upon extracellular calcium concentrations. Concentration-response studies demonstrated that the addition of up to 1 mM EGTA to standard growth medium, with a more than 4000-fold decrease in extracellular calcium concentration, had no effect on the activity of amphotericin B against C. albicans. Amphotericin B did affect the kinetics of calcium influx acutely (< or =10 min), but had no net effect on long-term (1-24 h) calcium accumulation. Calcium efflux was also not affected by amphotericin B. These results indicate that, unlike its effects on mammalian cells, the toxicity of amphotericin B against C. albicans is not dependent upon increased movement of calcium across the cell membrane or the presence of extracellular calcium.     

Effect of amphotericin B, fluconazole and itraconazole on intracellular Candida albicans and germ tube development in macrophages

Candida albicans may resist intracellular killing by macrophages through the formation of germ tubes. Antifungal drugs that inhibit intracellular germ tube formation could therefore facilitate host defence against C. albicans. We assessed the effects of amphotericin B and the new triazole drugs fluconazole and itraconazole on the multiplication and intracellular germ tube formation of C. albicans phagocytosed by murine peritoneal macrophages, and compared the findings with the effects of these drugs on C. albicans in the absence of macrophages. The fungicidal effect of amphotericin B against C. albicans in macrophages was less prominent than that found for extracellular candida. However, amphotericin B completely blocked germ tube formation of C. albicans both in macrophages and extracellularly. Fluconazole and itraconazole had little effect on the number of candida but significantly, although incompletely, inhibited germ tube formation both inside macrophages and extracellularly. The inhibition of intracellular germ tube formation by the triazoles may facilitate host defences against C. albicans and contribute to the efficacies of these drugs in vivo.     

In vitro activity of A-192411.29, a novel antifungal lipopeptide

A-192411.29 is a novel antifungal agent derived from the structural template of the natural product echinocandin. The in vitro activity of A-192411.29 against common pathogenic yeasts was assessed by National Committee for Clinical Laboratory Standards method M27-A. It demonstrated broad-spectrum, fungicidal activity and was active against the most clinically relevant yeasts, such as Candida albicans, Candida tropicalis, and Candida glabrata, as well as less commonly encountered Candida species; in general, its potency on a weight basis was comparable to that of amphotericin B. It maintained potent in vitro activity against Candida strains with reduced susceptibilities to fluconazole and amphotericin B. The in vitro activity of A-192411.29 against Cryptococcus neoformans was comparable to its activity against Candida spp. However, A-192411.29 did not demonstrate complete growth inhibition of Aspergillus fumigatus by the broth microdilution method used. A-192411.29 possesses an antifungal profile comparable to or better than those of fluconazole and amphotericin B against pathogenic yeasts, including strains resistant to fluconazole or amphotericin B, suggesting that it may be a therapeutically useful new antifungal drug.     

Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans.

Time-kill curves were determined for three isolates of Candida albicans tested against fluconazole and amphotericin B at multiples of the MIC. Fluconazole produced fungistatic activity, with concentration-related growth effects observed over a narrow range of concentrations. Amphotericin B exhibited fungicidal activity, with enhancement of activity over a broader range of concentrations.     

In Vitro Antifungal Activity and Cytotoxicity of a Novel Membrane-Active Peptide

In the present study, we investigated the antifungal activity and cytotoxicity of a novel membrane-active peptide, KKVVFKVKFKK (MP). MP inhibited the growth of various pathogenic fungi isolated from patients and of fluconazole-resistant fungi at concentrations of 2 to 32 microg/ml. MP had potent fungicidal activity; the minimal fungicidal concentrations of the peptide were no more than fourfold greater than the MICs. Time course experiments of MP-induced killing of Candida albicans ATCC 36232 showed that the rate of killing was rapid and depended on the concentration of MP. MP had a strong synergism with other antifungal drugs; the fractional inhibitory concentration index values of MP with amphotericin B and fluconazole for C. albicans were 0.16 and 0.02, respectively. The 50% inhibitory concentrations of MP for NIH 3T3 and Jurkat cells were approximately 100 times higher than the MIC for C. albicans ATCC 36232, indicating that MP had high selectivity between the fungal and mammalian cells. These results suggest that MP has great advantages in the development of antifungal agents.     

Multiple effects of green tea catechin on the antifungal activity of antimycotics against Candida albicans

OBJECTIVES: The susceptibility of Candida albicans to catechin under varying pH conditions and the synergism of the combination of catechin and antimycotics were evaluated.Method: Antifungal activity was determined by broth dilution and calculation of cfu. RESULTS: The antifungal activity of catechin was pH dependent. The concentration of epigallocatechin gallate (EGCg) causing 90% growth inhibition of tested strains of C. albicans was 2000 mg/L at pH 6.0, 500-1000 mg/L at pH 6.5 and 15.6-250 mg/L at pH 7.0. Among catechins, pyrogallol catechin showed stronger antifungal activity against C. albicans than catechol catechin. The addition of 6.25-25 or 3.12-12.5 mg/L EGCg to amphotericin B 0.125 or 0.25 mg/L (below MIC) at pH 7.0 resulted in enhancement, respectively, of the antifungal effect of amphotericin B against amphotericin B-susceptible or -resistant C. albicans. Combined treatment with 3.12-12.5 mg/L EGCg plus amphotericin B 0.5 mg/L (below MIC) markedly decreased the growth of amphotericin B-resistant C. albicans. When fluconazole-susceptible C. albicans was treated with 25-50 mg/L EGCg and fluconazole 0.125-0.25 mg/L (below MIC), its growth was inhibited by 93.0%-99.4% compared with its growth in the presence of fluconazole alone. The combined use of 12.5 mg/L EGCg and fluconazole 10-50 mg/L (below MIC) inhibited the growth of fluconazole-resistant C. albicans by 98.5%-99.7%. CONCLUSIONS: These results indicate that EGCg enhances the antifungal effect of amphotericin B or fluconazole against antimycotic-susceptible and -resistant C. albicans. Combined treatment with catechin allows the use of lower doses of antimycotics and induces multiple antifungal effects. It is hoped that this may help to avoid the side effects of antimycotics.     

Pharmacodynamics of fluconazole, itraconazole, and amphotericin B against Candida albicans

The objective of this study was to evaluate the pharmacodynamic activity of fluconazole, itraconazole, and amphotericin B against Candida albicans. Susceptibilities were determined according to the NCCLS guidelines (M27). Time-kill studies were performed using antifungal concentrations of 0.25-32 x MIC. Samples were withdrawn at predetermined timepoints, then plated using a spiral plater. Colony counts were determined after incubation at 35 degrees C for 24 h. The AUKC(0-48) was plotted against the concentration/MIC ratio. Candida isolates (95-2672, 96-15, and 95-2542) were classified as susceptible, susceptible-dose dependent, and resistant to fluconazole and itraconazole (MIC = 0.25 and 0.03 microg/mL, 32 and 0.5 microg/mL, 64 and 1 microg/mL; respectively). All three isolates were susceptible to amphotericin B (MIC = 0.13 microg/mL). Fluconazole inhibited the growth of the susceptible and S-DD isolates and was ineffective at all concentrations against the resistant isolate. Itraconazole, on the other hand, inhibited growth of the susceptible isolate, but was ineffective for the S-DD and resistant isolates. Maximal effectiveness was noted at the concentration 8 x MIC and 2 x MIC for fluconazole and itraconazole, respectively. Amphotericin B demonstrated concentration-dependent antifungal activity. The times necessary for the colony counts to fall below the limit of quantification were inversely related to increasing concentrations of amphotericin B. The maximal effect for amphotericin B was recorded at 2 x MIC. In summary, the triazoles inhibit growth of susceptible C. albicans; however, careful consideration should be given to the MIC for S-DD isolates because itraconazole may not be active if the MIC is reported in the higher susceptible-dose dependency range. In reference to amphotericin B, optimal activity may be achieved by maximizing the peak drug concentration/MIC ratio.     

Assessment of Antifungal Activities of Fluconazole and Amphotericin B Administered Alone and in Combination against Candida albicans by Using a Dynamic In Vitro Mycotic Infection Model

We evaluated the pharmacodynamic activities of fluconazole and amphotericin B given alone and in combination against Candida albicans by using an in vitro model of bloodstream infection that simulates human serum pharmacokinetic parameters for these antifungals. Fluconazole was administered as a bolus into the model to simulate regimens of 200 mg every 24 h (q24h) and 400 mg q24h. Amphotericin B was administered at doses producing the peak concentration (2.4 μg/ml) observed with a regimen of 1 mg/kg of body weight q24h. A combination regimen of fluconazole (400 mg q24h) and amphotericin B (1 mg/kg q24h) administered simultaneously and as a staggered regimen (amphotericin B bolus given 8 h after fluconazole bolus) was also simulated in the model to characterize possible antagonism between these agents. Fluconazole alone and amphotericin B alone demonstrated fungistatic (<99.9% reduction in numbers of CFU per milliliter from the starting inoculum) and fungicidal (>99.9% reduction) activity, respectively. When fluconazole and amphotericin B were administered simultaneously, fungicidal activity similar to that observed with amphotericin B alone was observed. Staggered administration of fluconazole and amphotericin B, however, resulted in a substantial reduction of the fungicidal activity of amphotericin B, producing fungistatic activity similar to that observed with noncombination fluconazole regimens. These results demonstrate the usefulness of this model for comparing the in vitro pharmacodynamic characteristics of different antifungal regimens and support the theory of azole-polyene antagonism. The effects of this antagonism on the in vivo activity and clinical usefulness of combination antifungal therapy, however, remain to be determined.     

Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro.     

In vitro activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole compared with those of amphotericin B and fluconazole against clinical isolates of Candida spp. and fluconazole-resistant Candida albicans

OBJECTIVES: The aim of this study was to investigate the in vitro antifungal activity of an isothiosemicarbazone cyclic analogue against isolates of Candida spp. including fluconazole-resistant Candida albicans. METHODS: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of Candida spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against C. albicans biofilms was also investigated. Toxicity in vitro was evaluated by MTT reduction assay. RESULTS: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against C. albicans and Candida krusei at concentrations lower than those shown by amphotericin B and fluconazole (P < 0.05). It maintained potent in vitro activity against fluconazole-resistant C. albicans isolates. Fungicidal activity occurred at concentrations 1-2 doubling dilutions greater than the corresponding MICs, and time-kill analysis indicated that a 99.9% loss of C. albicans viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of C. albicans ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC50 of amphotericin B was observed. CONCLUSIONS: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.     

Rates and Extents of Antifungal Activities of Amphotericin B, Flucytosine, Fluconazole, and Voriconazole against Candida lusitaniae Determined by Microdilution, Etest, and Time-Kill Methods

Amphotericin B, flucytosine, fluconazole, and voriconazole alone and in combination were evaluated against isolates of Candida lusitaniae. MICs were determined by broth microdilution and Etest, and time-kill studies were conducted. Amphotericin B resulted in fungicidal activity against most isolates, whereas fluconazole, voriconazole, and flucytosine produced primarily fungistatic activities. The addition of flucytosine to amphotericin B resulted in a faster rate and greater extent of kill for isolates for which the MICs of amphotericin B were elevated.     

Comparison of fluconazole and amphotericin B for treatment of experimental Candida endocarditis caused by non-C. albicans strains.

Amphotericin B and fluconazole were compared for the treatment of experimental Candida endocarditis caused by Candida tropicalis and C. parapsilosis. Rabbits received no therapy, amphotericin B (1 mg/kg of body weight per day intravenously), or fluconazole (100 mg/kg/day intraperitoneally) for either 11 or 21 days. Against both species, amphotericin B and fluconazole were equally effective overall; however, amphotericin B was more rapidly fungicidal than fluconazole in vivo against C. tropicalis.     

Novel aspect of amphotericin B action: accumulation in human monocytes potentiates killing of phagocytosed Candida albicans.

The influence of low doses of amphotericin B on the capacity of human monocytes to kill Candida albicans was investigated. Killing rates were quantified by a novel flow cytometric assay and were found to be 37% +/- 3% (standard error of the mean) after 3 h. Preincubation of monocytes for 6 to 20 h with low concentrations of amphotericin B (0.2 microgram/ml) resulted in a markedly augmented fungicidal capacity. Enhancement of killing was 80% +/- 11% (standard error of the mean) over that by the controls. This effect did not appear to be due to amphotericin B-dependent monocyte activation; the respiratory burst and expression of human leukocyte antigen-DR were unaltered, and no stimulation of interleukin-1 beta release occurred. Cell-associated amphotericin B was extracted with acetonitrile and was quantified by scanning spectrophotometry. Amphotericin B appeared to accumulate in the cells, and intracellular concentrations attained after overnight incubation in 1 microgram of the drug per ml were estimated to be in the range of 50 fg per cell. The fact that intracellular accumulation was responsible for the enhanced fungicidal capacity of monocytes was supported by the findings that killing of Staphylococcus aureus remained normal and enhancement of killing of an amphotericin B-resistant C. albicans strain was minimal. Dramatic enhancement of monocyte fungicidal capacity probably extends to other amphotericin B-susceptible fungi and could represent a hitherto unrecognized determinant underlying the curative properties and prophylactic efficacy of this drug.     

Influence of Human Serum on Antifungal Pharmacodynamics with Candida albicans

Antifungal susceptibilities (NCCLS, approved standard M27-A, 1997) were determined for the reference strain ATCC 90028 and 21 clinical isolates of Candida albicans with varying levels of fluconazole susceptibility using RPMI 1640 (RPMI) and 80% fresh human serum-20% RPMI (serum). Sixty-four percent (14 of 22) of the isolates tested demonstrated significant decreases (> or = 4-fold) in fluconazole MICs in the presence of serum, and the remaining eight isolates exhibited no change. Itraconazole and ketoconazole, two highly protein-bound antifungal agents, had MICs in serum that were increased or unchanged for 46% (10 of 22) and 41% (9 of 22) of the isolates, respectively. All 10 isolates tested against an investigational antifungal agent, LY303366, demonstrated significant increases in the MIC required in serum, while differences in amphotericin B MICs in the two media were not observed. Four of 10 isolates tested demonstrated fourfold higher flucytosine MICs in serum than in RPMI. Postantifungal effects (PAFEs) and 24-h kill curves were determined by standard methods for selected isolates. At the MIC, fluconazole, itraconazole, ketoconazole, flucytosine, and LY303366 kill curves and PAFEs in RPMI were similar to those in serum. Isolates of fluconazole-resistant C. albicans required lower MICs in serum than in RPMI, without relative increases in fungal killing or PAFEs. Isolates tested against amphotericin B demonstrated significantly reduced killing and shorter PAFEs in serum than in RPMI without observable changes in MIC. In conclusion, antifungal pharmacodynamics in RPMI did not consistently predict antifungal activity in serum for azoles and amphotericin B. Generally speaking, antifungal agents with high protein binding exhibited some form of reduced activity (MIC, killing, or PAFE) in the presence of serum compared to those with low protein binding.     

Voriconazole-induced inhibition of the fungicidal activity of amphotericin B in Candida strains with reduced susceptibility to voriconazole: an effect not predicted by the MIC value alone

An antagonistic effect of voriconazole on the fungicidal activity of sequential doses of amphotericin B has previously been demonstrated in Candida albicans strains susceptible to voriconazole. Because treatment failure and the need to switch to other antifungals are expected to occur more often in infections that are caused by resistant strains, it was of interest to study whether the antagonistic effect was still seen in Candida strains with reduced susceptibility to voriconazole. With the hypothesis that antagonism will not occur in voriconazole-resistant strains, C. albicans strains with characterized mechanisms of resistance against voriconazole, as well as Candida glabrata and Candida krusei strains with differences in their degrees of susceptibility to voriconazole were exposed to voriconazole or amphotericin B alone, to both drugs simultaneously, or to voriconazole followed by amphotericin B in an in vitro kinetic model. Amphotericin B administered alone or simultaneously with voriconazole resulted in fungicidal activity. When amphotericin B was administered after voriconazole, its activity was reduced (median reduction, 61%; range, 9 to 94%). Levels of voriconazole-dependent inhibition of amphotericin B activity differed significantly among the strains but were not correlated with the MIC values (correlation coefficient, -0.19; P = 0.65). Inhibition was found in C. albicans strains with increases in CDR1 and CDR2 expression but not in the strain with an increase in MDR1 expression. In summary, decreased susceptibility to voriconazole does not abolish voriconazole-dependent inhibition of the fungicidal activity of amphotericin B in voriconazole-resistant Candida strains. The degree of interaction could not be predicted by the MIC value alone.     

Comparison of the efficacies of amphotericin B, fluconazole, and itraconazole against a systemic Candida albicans infection in normal and neutropenic mice.

We compared the efficacies of the new triazole antifungal drugs fluconazole and itraconazole with that of amphotericin B in vitro and in an animal model of systemic candidiasis in normal and neutropenic mice. Antifungal treatment with fluconazole (2.5 to 20 mg/kg orally twice daily), itraconazole (10 to 40 mg/kg orally twice daily), or amphotericin B (0.1 to 4 mg/kg intraperitoneally once daily) was started 1 day after intravenous injection of 10(4) Candida albicans into normal mice or 10(3) C. albicans into neutropenic mice; the drugs were administered for 2 days. In normal mice the efficacy of treatment, which was assessed on the basis of the number of C. albicans cultured from the kidney, was greater for amphotericin B than for the triazoles. Fluconazole was more potent than itraconazole on the basis of equivalent doses, although itraconazole was more potent on the basis of the amount of free drug that was available. In neutropenic mice amphotericin B was less effective than it was in normal mice, whereas the triazoles were equally effective in normal and neutropenic mice. This was not expected, since in vitro data showed that amphotericin B was highly fungicidal, whereas both fluconazole and itraconazole had only a minimal effect on the growth of C. albicans in vitro.     

In vitro interaction of fluconazole and amphotericin B administered sequentially against Candida albicans: effect of concentration and exposure time

This study investigated the potential antagonism of fluconazole on amphotericin B activity against Candida albicans when administered sequentially in vitro. Yeast cells were exposed to fluconazole for time periods ranging from 0 to 24 h before the addition of amphotericin B. The combination showed fungicidal (≥3 log₁₀ reduction in CFU/mL) activity. After 4 h of exposure to fluconazole, amphotericin B activity was partially inhibited at the lower concentration tested (0.25 × MIC). Amphotericin B activity was dramatically decreased by previous exposure to fluconazole for greater than or equal to 8 h at both the high and low concentrations tested. The activity of amphotericin B against yeast exposed to fluconazole for at least 8 h was indistinguishable from fluconazole alone and was fungistatic (≤2 log₁₀ reduction in CFU/mL). This inhibition of amphotericin B activity persisted for a very short period (<6 h) after removal of fluconazole from the culture medium, indicating the need for continued exposure to fluconazole for lasting inhibition of amphotericin B activity.     

In-vitro activity of cilofungin (LY121019) in comparison with amphotericin B

The in-vitro activity of cilofungin, a derivative of echinocandin B, was compared with that of amphotericin B in Sabouraud dextrose and Antibiotic Medium No. 3 against 100 clinical isolates of yeasts. Cilofungin appeared to be as effective, as amphotericin B against Candida albicans and yet more effective against Can. tropicalis as far as growth inhibition was concerned. Cilofungin was less active than amphotericin B against Can. (Torulopsis) glabrata and other species of Candida. It was not active against Cryptococcus neoformans. Minimum fungicidal concentrations (MFCs) of cilofungin were highly dependent on the medium, especially with Can. albicans. Low MFCs were observed in Antibiotic Medium 3 and very high MFCs were measured in Sabouraud's medium. Using a killing curve method, the initial rate of killing of amphotericin B was proportional to concentration with Can. albicans, Can. tropicalis, and Can. glabrata. With cilofungin the rate of killing was proportional to concentration only for Can. tropicalis.     

Alternatives to amphotericin B for Candida rugosa infection

OBJECTIVE: Amphotericin B failure is frequently seen in patients with candidaemia caused by Candida rugosa. We evaluated amphotericin B, fluconazole, posaconazole and voriconazole as alternative treatments against infection in mice with two isolates of C. rugosa. METHODS: Neutropenic mice were inoculated intravenously with C. rugosa. Amphotericin B, fluconazole, posaconazole and voriconazole were administered for 7 days after infection. Efficacy of the antifungal treatment was assessed by survival and tissue burden of C. rugosa. RESULTS: All of the four drugs significantly prolonged survival over controls. With both isolates, kidney counts were reduced significantly below controls for amphotericin B, fluconazole and posaconazole. However, voriconazole was less effective than the other antifungals. CONCLUSION: Despite poor clinical response to amphotericin B, in vivo data indicate that amphotericin B increases organ clearance and survival over untreated controls. However, although voriconazole improved survival over controls, increased tissue clearance was not seen. This discrepancy may be caused by rapid clearance of voriconazole in mice. These studies suggest fluconazole, posaconazole or voriconazole may be useful alternatives to amphotericin B in therapy of C. rugosa infection.     

A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology

This study evaluated the in vitro pharmacodynamics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans. MICs were determined for three clinical isolates according to NCCLS guidelines (M27). Time-kill studies were performed using antifungal concentrations of 0.25-32 x MIC and inocula of 10(3) and 10(5) CFU/ml. At predetermined time points over 72 hours, samples of each inoculum/drug combination were withdrawn and plated using a spiral plater. Colony counts were determined after incubation at 35 degrees C for 48 hours. Area under the kill curves (AUKCs) were plotted versus the AUC/MIC ratios. Inoculum effect was evaluated by calculating an estimated AUKC for the low inoculum then comparing it to the measured low inoculum using the unpaired Student's t-test. The MICs of fluconazole and itraconazole for isolate 97-1199, 97-1061, and 97-585 were 2, 4, 32 microg/ml and 0.03, 0.06, 0. 5 microg/ml, respectively. For amphotericin B, the MIC was 0. 25 microg/ml for each isolate. The triazoles demonstrated fungistatic activity against each isolate at both inocula with the exception of itraconazole against C. neoformans 97-585. Maximal suppression was noted at concentrations 8-16 x MIC correlating with an AUC/MIC of 192 for both inocula. Conversely, amphotericin B was fungicidal and displayed concentration-dependent activity against each isolate at both inocula. Maximal killing was observed at concentrations >4 x MIC for the low inoculum and >8 x MIC for the high inoculum for each isolate. No statistically significant differences were detected between the measured and estimated AUKCs for each antifungal agent. In conclusion, our results suggest that the triazoles were most effective against C. neoformans when concentrations were maintained at 8-16 x MIC. Amphotericin B, on the other hand, was concentration-dependent; thus, greater activity was exerted at higher concentrations.