In Vitro and In Vivo Evaluation of Synergistic Effects of Everolimus in Combination with Antifungal Agents on Exophiala dermatitidis

Abstract

To investigate the combined function of the novel oral mTOR inhibitor, everolimus, with antifungal agents and their potential mechanisms against Exophiala dermatitidis, the CLSI microliquid-based dilution method M38-A2, chequerboard technique, and disk diffusion testing were performed. The efficacy of everolimus was evaluated in combination with itraconazole, voriconazole, posaconazole, and amphotericin B against 16 clinically isolated strains of E. dermatitidis. The synergistic effect was determined by measuring the MIC and fractional inhibitory concentration index. Dihydrorhodamine 123 was used for the quantification of ROS levels. The differences in the expression of antifungal susceptibility-associated genes were analyzed following different types of treatment. Galleria mellonella was used as the in vivo model. While everolimus alone showed minimal antifungal effects, combinations with itraconazole, voriconazole, posaconazole, or amphotericin B resulted in synergy in 13/16 (81.25%), 2/16 (12.5%), 14/16 (87.75%), and 5/16 (31.25%) of isolates, respectively. The disk diffusion assay revealed that the combination of everolimus and antifungal drugs showed no significant increase in the inhibition zones compared with the single agent, but no antagonistic effects were observed. Combination of everolimus and antifungal agents resulted in increased ROS activity (everolimus + posaconazole versus posaconazole [P < 0.05], everolimus + amphotericin B versus amphotericin B [P < 0.002]). Simultaneously, compared to mono-treatment, the combination of everolimus + itraconazole suppressed the expression of MDR2 (P < 0.05) and the combination of everolimus + amphotericin B suppressed the expression of MDR3 (P < 0.05) and CDR1B (P < 0.02). In vivo, combinations of everolimus and antifungal agents improved survival rates, particularly the combination of everolimus + amphotericin B (P < 0.05). In summary, the in vivo and in vitro experiments performed in our study suggest that the combination of everolimus with azoles or amphotericin B can have synergistic effects against E. dermatitidis, potentially due to the induction of ROS activity and inhibition of efflux pumps, providing a promising new approach for the treatment of E. dermatitidis infections. IMPORTANCE Cancer patients with E. dermatitidis infection have high mortality if untreated. Clinically, the conventional treatment of E. dermatitidis is poor due to the long-term use of antifungal drugs. In this study, we have for the first time investigated the interaction and action mechanism of everolimus combined with itraconazole, voriconazole, posaconazole, and amphotericin B on E. dermatitidis in vitro and in vivo, which provided new ideas and direction for further exploring the mechanism of drug combination and clinical treatment of E. dermatitidis.

Keywords: E. dermatitidis; ROS; antifungal agents; combination; efflux pumps; everolimus.

FIG 1

ROS activity level changes in the single and combined groups. The proportion of DHR-123 oxidized by ROS in the control and experimental groups and related statistical analysis. (A) Changes in ROS generation ratio across the single and combined drug groups. In flow cytometry analysis, the abscissa represents the relative fluorescence intensity, and the ordinate represents the spore count. The dark blue area represents the percentage of spores (P3) with emitted fluorescence after DHR-123 was oxidized. The peak value in the dark blue area indicates the largest number of oxidized spores under the corresponding fluorescence intensity. (B) The ROS activity levels were significantly increased in the group treated with EVL plus POS or AMB compared to POS or AMB alone group, respectively. Dihydrorhodamine 123, DHR-123; EVL, everolimus; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B. *, P < 0.05; **, P < 0.01; ns, no significance.

FIG 2

The results of antifungal sensitivity related gene RT-qPCR in the single and combined groups. Note: Total RNA comparison of different genes in drug-free and single-drug groups (A), and total RNA comparison of different genes in single-drug and combination groups (B). EVL, everolimus; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B. *, P < 0.05; **, P < 0.015; ns, no significance.

FIG 3

The results of disk diffusion testing of everolimus combined with antifungal agents. Note: Disk diffusion test: E. dermatitidis BMU00028 statistical analysis of antifungal agents alone and in combination with everolimus (A). Record the diameter of the inhibition area. The blank tray was impregnated with EVL, ITC, VOR, POS, AMB, EVL + ITC, EVL + VRC, EVL + POS, EVL + AMB, and saline controls (B). EVL, everolimus; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B; ns, no significance.

FIG 4

Survival rates in mono-treatment and combination treatment groups. Note: The survival curves of larvae infected with E. dermatitidis after different interventions. The control groups of uninfected group and saline group showed all survival. The uninfected and saline controls demonstrated 100% survival at 6 days. Azoles alone and AMB or AMB combine with EVL can significantly improve the survival rate of larvae. EVL, everolimus; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B; *, P < 0.05; ***, P ≤ 0.0005; ****, P < 0.0001; ns, no significance.