In vitro and in vivo antimicrobial effects of domiphen combined with itraconazole against Aspergillus fumigatus

Abstract

Aspergillus fumigatus, a prevalent saprophytic fungus in the atmosphere, is known to rapidly induce severe invasive aspergillosis (IA) upon inhalation of its conidia by humans or animals. The mortality rate associated with IA exceeds 50%. The misuse of antifungal agents has contributed to the emergence of numerous highly pathogenic drug-resistant strains of A. fumigatus. Our study found that the combination of domiphen and itraconazole had sound synergistic antimicrobial effects against wild-type and itraconazole-resistant A. fumigatus in vivo and in vitro through MIC, FIC, plate inoculation, growth curve experiments, and Galleria mellonella infection model. Drug cytotoxicity and pharmacological tests for acute toxicity assays demonstrated that both itraconazole and domiphen showed minimal cytotoxicity and good biocompatibility. The transcriptome sequencing experiment demonstrated that domiphen exerted a suppressive effect on the expression of various genes, including those involved in drug efflux, redox regulation, and cellular membrane and cell wall remodeling. The present investigation explores the synergistic antimicrobial mechanisms of domiphen and itraconazole, encompassing three key aspects: (i) domiphen inhibited the efflux of itraconazole by reducing the expression of drug efflux-related genes, (ii) the combination has good ability to disrupt the cell membrane and cell wall, (iii) the combination also can remove biofilm more effectively. In summary, the utilization of domiphen as a synergist of itraconazole exhibited disruptive effects on the biofilm, cell wall, and cell membrane of A. fumigatus. This subsequently led to a modified distribution of itraconazole within the fungal organism, ultimately resulting in enhanced antifungal efficacy. The results of this study may provide a new therapeutic strategy for the treatment of IA caused by drug-resistant A. fumigatus.

Keywords: Aspergillus fumigatus; azole resistance; domiphen; itraconazole; synergistic effect.

Figure 1

Antimicrobial capacity and biocompatibility of the drug. (A) MICs of domiphen and itraconazole against four strains of Aspergillus fumigatus. In each group of pictures, the first two test tubes are clarified MM solutions, the last two test tubes are turbid MM solutions containing the mycelium of A. fumigatus. (B) Cytotoxicity of itraconazole and domiphen. The cells used were HK-2 (human renal cortex proximal convoluted tubule epithelial cells) and HT-22 (mouse hippocampus nerve cells). (C) Acute toxicity experiments with itraconazole and domiphen. Both drugs have low toxicity to zebrafishes.

Figure 2

In vitro synergistic antimicrobial activity of drug combinations against Aspergillus fumigatus. (A) Plate spotting of domiphen and itraconazole against four strains of A. fumigatus. The concentration of A. fumigatus in the first, second and third columns was 10⁸, 10⁷, and 10⁶ CFU/mL, respectively. (B) Size of colony diameter after drug inhibition. Combination therapy for A. fumigatus is more effective than monotherapy. D+I, Domiphen+Itraconazole. (C) Determination of the antimicrobial activity of domiphen and itraconazole against four A. fumigatus strains. “+” represents the administration of 1/2 × MIC concentration of the single drug; or 1/4 × MIC concentration of combined drugs. “−” represents administration of the same volume of sterile water. After 36 h of treatment, the number of CFU was measured. Data from six replicates were collected to obtain mean log CFU values. Statistical analysis was performed to determine significant differences between the combined treatments and the single compounds. ^***p < 0.001; ^****p < 0.0001 according to unpaired two-tailed t-test. (D) Growth curves of domiphen, itraconazole and the combination against four strains of A. fumigatus. D+I, Domiphen+Itraconazole.

Figure 3

In vivo effects of drug combinations on Aspergillus fumigatus antifungal activity. (A) Day 7 of an in vivo experiment with Galleria mellonella larvae. (a) injected with saline, (b) injected with itraconazole, (c) injected with domiphen, (d) injected with combination drug, (e) injected with fungus and combination drug, (f) injected with fungus and itraconazole, (g) injected with fungus and domiphen, (h) injected with fungus (Shjt 40). (B) Survival curve of G. mellonella larvae. I, Itraconazole; D, Domiphen; ^*p <  0.05; ^***p < 0.001; ns, p > 0.05 according to log rank analysis.

Figure 4

mRNA expression of Aspergillus fumigatus after domiphen treatment. The heatmap showing log₂FC of each genes was generated using MeV (https://webmev.tm4.org/) based on RNA-seq data. Wild-type conidia were collected and incubated in MM for 18 h at 37°C followed by 1 h of domiphen (2 μg/mL) incubation.

Figure 5

Effect of domiphen and itraconazole on intracellular drug retention and biofilm of Aspergillus fumigatus. (A) Extracellular itraconazole content in A. fumigatus cells treated with itraconazole before and after domiphen treatment indicated as Itz and I+D, respectively. ^*p < 0.05; ^**p < 0.01 according to unpaired two-tailed t-test. (B) Biofilm clearance efficiency of domiphen, itraconazole, and domiphen combined with itraconazole against four A. fumigatus strains.

Figure 6

Effect of domiphen and itraconazole on cell membrane of Aspergillus fumigatus. (A) Propidium Iodide (PI) reagent staining of A. fumigatus cells with or without indicated drug treatment. D+I, Domiphen+Itraconazole. (B) Fluorescence intensity of drug-treated A. fumigatus. D+I, Domiphen+Itraconazole; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001. (C) Transmission Electron Microscopy (TEM) of A. fumigatus without (left) or with (right) drug treatment. Scale bar = 500 nm. (D) Cell wall thickness of A. fumigatus with and without combined drug treatment. D+I, Domiphen+Itraconazole; ^*p < 0.05.

Figure 7

Mechanism of synergistic antimicrobial of domiphen and itraconazole.