The Synergistic Effect of Tacrolimus (FK506) or Everolimus and Azoles Against Scedosporium and Lomentospora Species In Vivo and In Vitro

Abstract

Scedosporium and Lomentospora infections in humans are generally chronic and stubborn. The use of azoles alone cannot usually inhibit the growth of these fungi. To further explore the combined effect of multiple drugs and potential mechanisms of action, we tested the antifungal effects of tacrolimus (FK506) and everolimus in combination with azoles in vitro and in vivo on 15 clinical strains of Scedosporium/Lomentospora species and detected the level of Rhodamine 6G, ROS activity, and apoptosis. The in vitro results showed that the combinations of tacrolimus with itraconazole, voriconazole, and posaconazole showed synergistic effects on 9 strains (60%), 10 strains (73%), and 7 strains (47%), respectively, and the combinations of everolimus with itraconazole, voriconazole, and posaconazole showed synergistic effects on 8 strains (53%), 8 strains (53%), and 7 strains (47%), respectively. The synergistic effects might correspond to the elevated ROS activity (the tacrolimus + itraconazole group compared to the itraconazole group, (P < 0.05)), early apoptosis (itraconazole (P < 0.05) and voriconazole (P < 0.05) combined with everolimus), and late apoptosis (the tacrolimus + itraconazole group compared to the itraconazole group, (P < 0.01); the tacrolimus + posaconazole group compared to the posaconazole group, (P < 0.05)), but not inhibition of efflux pump activity. Our in vitro results suggested that a combination of tacrolimus or everolimus and azoles have a synergistic effect against Scedosporium/Lomentospora. The synergistic mechanisms of action might be triggering excessive ROS activity and apoptosis. In vivo, the survival rate of G. mellonella (sixth instar larvae) was significantly improved by tacrolimus alone, everolimus alone, azoles alone, and tacrolimus and everolimus combined with azoles separately (P < 0.05 for the tacrolimus group; P < 0.01 for the everolimus group and the itraconazole group; P = 0.0001 for the tacrolimus and posaconazole group; P < 0.0001 for other groups except the everolimus and itraconazole group, everolimus and posaconazole group, and tacrolimus and itraconazole group). From the results, we infer that the combination of tacrolimus or everolimus with azoles has obvious synergistic effect on Scedosporium/Lomentospora, and might enhance the level of apoptosis and necrosis. However, the synergistic effects were not related to the efflux pump. In conclusion, from our in vitro and in vivo study, tacrolimus and everolimus combined with azoles may have a synergistic effect in the treatment against Scedosporium/Lomentospora, improving the drug activity of azoles and promoting a better prognosis for patients.

Keywords: Azoles; Everolimus; Immunocompromised adults; Scedosprium; Tacrolimus.

Figure 1

Schematic diagram of drug sensitivity. The colors of green to yellow represent the amount of fungal growth in the various drug combination concentrations. Green indicates good fungal growth, yellow indicates poor fungal growth. T, tacrolimus; E, everolimus; I, itraconazole; V, voriconazole; P, posaconazole; and the number after the letter represents the concentration (µg/ml).

Figure 2

Changes in ROS generation ratio across the single and combined drug groups. (A) The proportion of Dihydrorhodamine 123 oxidized by ROS in the control and experimental groups and related statistical analysis. (B) In flow cytometry analysis, the abscissa represents the relative fluorescence intensity and the ordinate represents the spore count. The brown area represents the percentage of spores (P3) with emitted fluorescence after Dihydrorhodamine 123 was oxidized to Rhodamine 123. The peak value in the brown area indicates the largest number of oxidized spores under the corresponding fluorescence intensity. TAC, tacrolimus; EVL, everolimus; ITR, itraconazole; VRC, voriconazole; POS, posaconazole. *P < 0.05; ns, no significance.

Figure 3

The evaluation of efflux pump function. The effect of TAC combined with azoles on the Rhodamine 6G outflow of S. apiospermum (L12E). The extracellular Rhodamine 6G concentration of S. apiospermum (L12E) was determined using spectrophotometry. Energy-dependent Rhodamine 6G efflux was quantified by adding glucose, azole, and TAC with Rhodamine 6G (10 mM) or Rhodamine 6G (10 mM) in glucose-free PBS and measuring the absorbance of the supernatant at 488 nm. TAC, tacrolimus; EVL, everolimus; ITR, itraconazole; VRC, voriconazole; POS, posaconazole. The values represent the mean and SD of three independent experiments.

Figure 4

The effect of drug treatment on apoptosis. (A) The proportion of spores undergoing apoptosis at different stages of apoptosis in the control group and the experimental group and related statistical analysis. (B) In flow cytometry analysis, Annexin-V-FITC and PI fluorescents emit different wavelengths of fluorescence in the early and late stages of apoptosis. The lower left quadrant represents living cells; the upper left quadrant represents early apoptotic cells; the upper right quadrant represents necrotic and late apoptotic cells. TAC, tacrolimus; EVL, everolimus; ITR, itraconazole; VRC, voriconazole; POS, posaconazole. *P < 0.05; **P < 0.01; ns, no significance.

Figure 5

The survival curves of larvae infected with Scedosporium after different interventions. (A) TAC group (B) EVL group. G. mellonella infected with Scedosporium. Tacrolimus, everolimus, and azoles alone or in combination with tacrolimus or everolimus can significantly improve the survival rate of larvae. Tacrolimus and everolimus combined with azoles showed a synergistic effect on Scedosporium infection. TAC, tacrolimus; EVL, everolimus; ITR, itraconazole; VRC, voriconazole; POS, posaconazole. *P < 0.05; **P < 0.01; ***P = 0.0001; ****P < 0.0001; ns, no significance.