Fungicidal Activity Of Amphotericin Research Articles

Assessment of Candida auris Response to Antifungal Drugs Using Time–Kill Assays and an Animal Model

Background Candida auris is an emerging nosocomial pathogen that is resistant to Fluconazole and variably susceptible to other systemic drug classes. Treatment with echinocandins has been recommended based on MICs in the susceptible range, but supporting in vivo data is lacking.Methods We tested the MIC of C. auris strains (n = 12) to fluconazole, voriconazole, posaconazole. anidulafungin, amphotericin B and flucytosine. Representative C. auris strains from Israel and South Africa, and a reference C. albicans strain were analysed using time–kill curve assays. Fungicidal activity was defined as reduction of ≥3 log from baseline CFU/ml. Response to caspofungin treatment was assessed in BALB/c mice immunosuppressed with cyclophosphamide and inoculated with 7 × 107C. auris cells by tail vein injection. Mice were treated from day +1 to day +7 with caspofungin (IP) at doses of 1 or 5 mg/kg and compared with sham-treated controls. Survival was assessed daily. Kaplan-Meier survival analyses were performed and treatment arms were compared using the log-rank test.ResultsDrug susceptibility results (MIC50 and MIC90) were: fluconazole, 64 and 128 mg/l; voriconazole, 0.5 and 24 mg/l; posaconazole, 0.5 and 27 mg/l; anidulafungin, 0.03 and 0.06 mg/l; amphotericin B, 2 and 8 mg/l; flucytosine, 0.3 and 1 mg/l. Time–kill curve analyses showed log reduction from baseline CFU concentration of −3.0 to −2.8 for fluconazole (MIC ×1), 5.6–6.1 for amphotericin B (MIC ×4) and −0.4 to −0.9 for caspofungin (MIC ×16), consistent with fungicidal activity of amphotericin B and weak fungistatic activity of caspofungin. In the mouse model, survival rate was similar with sham treatment (33%) and treatment with caspofungin 1 mg/kg/day (44%) and 5 mg/kg/day (22%), P = 0.7.Conclusion Despite generally low MIC, caspofungin has only mild fungistatic activity on C. auris and no effect on survival in a mouse infection model. Amphotericin B has fungicidal activity against C. auris.Disclosures All authors: No reported disclosures.

Enhancement of the Fungicidal Activity of Amphotericin B by Allicin: Effects on Intracellular Ergosterol Trafficking

In Saccharomyces cerevisiae, ergosterol was visible in the plasma membrane of untreated cells and was enriched in the vacuole membrane in response to amphotericin B (AmB) treatment at a non-lethal concentration. The simultaneous addition of allicin was inhibitory to AmB-induced ergosterol enrichment in the vacuole membrane, resulting in increased sensitivity of the organelles to the disruptive action of AmB. Allicin was also inhibitory to ergosterol enrichment in the vacuole membrane that was achieved by its external addition to cells. The combined fungicidal activity of AmB and allicin was suppressed together with suppression of vacuole membrane damage in cells where ergosterol had been fully enriched in the vacuole membrane. AmB caused direct disruptive damage of the isolated vacuoles, but the antibiotic was apparently less effective in disrupting the organelles that were isolated after ergosterol enrichment. These findings suggest that allicin enhances AmB-induced vacuole membrane damage by inhibiting ergosterol trafficking from the plasma membrane to the vacuole membrane.

Characterization of the inhibitory effect of voriconazole on the fungicidal activity of amphotericin B against Candida albicans in an in vitro kinetic model

The aim of the present investigation was to study and characterize the effect of voriconazole on the fungicidal activity of amphotericin B. Four strains of Candida albicans susceptible to voriconazole were exposed to voriconazole and amphotericin B, either alone, simultaneously or sequentially in an in vitro kinetic model. Bolus doses resulting in voriconazole and amphotericin B concentrations of 0.005-5 and 2.5 mg/L, respectively, were administered. Antifungal-containing RPMI 1640 was eliminated and replaced by a fresh medium using a peristaltic pump, with a flow rate adjusted to obtain the desired half-lives. With two drugs tested, a computer-controlled dosing pump compensated for differences in the elimination rates. Using static time-kill methodology, one C. albicans strain was exposed to 5 mg/L voriconazole for varying durations followed by 2.5 mg/L amphotericin B after three repeated washes of voriconazole. Voriconazole and amphotericin B treatment alone resulted in fungistatic and fungicidal activities, respectively. Simultaneous administration of voriconazole and amphotericin B resulted in fungicidal activity, whereas only fungistatic activity was observed when repeated doses of amphotericin B were administered sequentially after voriconazole at 24-96 h. The inhibition of the fungicidal activity of amphotericin B was voriconazole dose-dependent, but seemed to be recovered once the voriconazole concentration fell below the MIC. The fungicidal activity was quickly regained after the removal of voriconazole, irrespective of the duration of voriconazole pre-exposure. Voriconazole inhibited the fungicidal effect of sequentially administered amphotericin B in a concentration- and time-dependent manner; the clinical significance of this needs further investigation.

Effect of CpG DNA Oligodeoxynucleotides and Echinacea on the Fungicidal Activity of Amphotericin B in a Disseminated Murine Candidal Infection Model

Effect of CpG DNA Oligodeoxynucleotides and Echinacea on the Fungicidal Activity of Amphotericin B in a Disseminated Murine Candidal Infection Model

In-vitro interaction of terbinafine with amphotericin B, fluconazole and itraconazole against clinical isolates of Candida albicans.

A chequerboard titration broth microdilution method, performed according to the recommendations of the National Committee for Clinical Laboratory Standards, was applied to study the in-vitro interaction of terbinafine with amphotericin B, fluconazole and itraconazole against 30 strains of Candida albicans isolated from the oral cavities of AIDS patients. MICs were determined spectrophotometrically at 490 nm and read at either 24 h or 48 h. The end-point was defined as the drug concentration resulting in > or = 90% inhibition of growth relative to control growth. Synergy, defined as a fractional inhibitory concentration (FIC) index of < or = 0.50, was observed in 93% (28 of 30) of terbinafine-amphotericin B interactions, in 47% (14 of 30) of terbinafine-fluconazole interactions and in 43% (13 of 30) of terbinafine-itraconazole interactions; antagonism (FIC > 2.0) was not observed. Where synergy was not achieved, there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination. Reading the MICs on day 2 did not significantly affect the mode of interaction of terbinafine-triazoles, while for terbinafine-amphotericin B the proportion of synergic interactions dropped from 93% (28 of 30) to 30% (nine of 30; P = 0.0001). Antagonism was not observed for any drug combination even at 48 h. Minimum fungicidal concentrations (MFCs) of all drugs alone and in combination were determined against five isolates. Neither terbinafine nor the two triazoles showed fungicidal activity when tested alone or in combination. The fungicidal activity of amphotericin B was slightly enhanced when combined with terbinafine, there being a decrease of two-fold dilutions in the amphotericin B MFCs against all five isolates tested. Thus terbinafine enhances the activities of amphotericin B and triazoles against C. albicans in vitro. Clearly, clinical studies are warranted to elucidate further the potential utility of these combination therapies.

Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum

This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 micrograms/ml, respectively, for up to 10 h. Hence, these agents were not fungistatic when tested in the presence of serum. (ii) Simultaneous application of 5-fluorocytosine had neither enhancing nor inhibitory effects on the fungicidal activity of amphotericin B. However, yeasts that were preincubated for 20 h with 5-fluorocytosine became less susceptible to killing by amphotericin B. (iii) Fluconazole exerted a frank antagonistic effect on the fungicidal activity of amphotericin B. Thus, under our in vitro conditions, both fluconazole and 5-fluorocytosine can overtly antagonize the candidacidal action of amphotericin B.