Allicin as a Volatile or Nebulisable Antimycotic for the Treatment of Pulmonary Mycoses: In Vitro Studies Using a Lung Flow Test Rig

Abstract

Fungal infections of the lung are an increasing problem worldwide and the search for novel therapeutic agents is a current challenge due to emerging resistance to current antimycotics. The volatile defence substance allicin is formed naturally by freshly injured garlic plants and exhibits broad antimicrobial potency. Chemically synthesised allicin was active against selected fungi upon direct contact and via the gas phase at comparable concentrations to the pharmaceutically used antimycotic amphotericin B. We investigated the suppression of fungal growth by allicin vapour and aerosols in vitro in a test rig at air flow conditions mimicking the human lung. The effect of allicin via the gas phase was enhanced by ethanol. Our results suggest that allicin is a potential candidate for development for use in antifungal therapy for lung and upper respiratory tract infections.

Keywords: Paecilomyces; allicin; antimycotic; lung infection; mycosis.

Figure A1

Macroscopic and microscopic characteristics of Penicillium crustosum. Growth was studied on different culture media (Appendix A). A 20 µL spore suspension (5 × 10⁵ spores per mL) was pipetted three times onto different solid media and incubated at 21 °C for five days. (A) Surface growth and (B) colony reverse were documented photographically. Using transmitted light microscopy (light microscope DMRBE, Leica, Wetzlar, Germany), hyphal growth, (C) conidiophore structure and (D) spores of Penicillium crustosum were examined.

Figure A2

Macroscopic and microscopic characteristics of Paecilomyces formosus. Growth was studied on different culture media (Appendix A). A 20 µL spore suspension (5 × 10⁵ spores per mL) was pipetted three times onto different solid media and incubated at 37 °C for five days. (A) Surface growth and (B) colony reverse were documented photographically. Using transmitted light microscopy (light microscope DMRBE, Leica, Wetzlar, Germany), (C) hyphal growth, (D) conidiophore structure and (E) spores of Paecilomyces formosus were examined.

Figure A3

Macroscopic and microscopic characteristics of Cladosporium cladosporioides. Growth was studied on different culture media (A). A 20 µL spore suspension (5 × 10⁵ spores per mL) was pipetted three times onto different solid media and incubated at 21 °C for five days. (A) Surface growth and (B) colony reverse were documented photographically. Using transmitted light microscopy (light microscope DMRBE, Leica, Wetzlar, Germany), hyphal growth, (C) conidiophore structure and (D,E) spores of Cladosporium cladosporioides were examined.

Figure 1

Allicin inhibits spore germination upon direct contact in a concentration-dependent manner. (A) Fungal spores (10⁶ spores per mL) were added to 20 mL PDA medium (50 °C). After solidification, three holes (Ø = 0.6 cm) were punched into the agar and filled with 40 µL of 10 µM, 20 µM aqueous allicin solution or deionised water (diH₂O), respectively. Triplicates were made for each fungus and incubated for 48 h. (B) n = 3, error bars show standard deviation. Same letter indicates no significant difference (p > 0.05) in a One-Way RM ANOVA using the Holm–Šidák method.

Figure 2

Allicin inhibits spore germination via the gas phase. (A) Fungal spores (10⁶ spores per mL) were added to 20 mL PDA medium (50 °C). After the medium solidified, diH₂O, 96% ethanol or 50 mM aqueous allicin solution were applied to the Petri dish lid. Triplicates of each fungal sample were prepared and incubated for 48 h. (B) n = 3, error bars show standard deviation. Same letter indicates no significant difference (p > 0.05) in a One-Way RM ANOVA using the Holm–Šidák method.

Figure 3

Allicin efficacy is comparable to that of amphotericin B. Fungal spore suspensions were mixed with sample solution. Spore germination was examined microscopically after 48 h of incubation. (A) Germinated and ungerminated spores were counted in an image field and the ratio of germinated spores to total spore number was calculated (diH₂O or 3% DMSO was set to 100% spore germination). The mean values of three biological replicates with three technical replicates each are shown. Sample solution concentration was plotted logarithmically. Error bars represent the standard deviation. (B) n = 9, error bars show standard deviation. Same letter indicates no significant difference (p > 0.05) in a One-Way RM ANOVA using the Holm–Šidák method.

Figure 4

Concentration-dependent inhibition of spore germination after aerosol treatment of the lung model with aqueous allicin solution. A volume of 20 mL of 2% (w/v) PDA medium (50 °C) mixed with spore suspension of Paecilomyces formosus (10⁶ spores per mL) was used to coat each lung model half-shell. The supply air was adjusted to 6 L per min. The model was treated with aqueous allicin solution in the lung simulation apparatus for 20 min and subsequently incubated at 37 °C. (A) After 24 h and 48 h of incubation, fungal growth was detected by staining with MTT. The lower half of the experiment model is shown. (B) Binary images of the 48 h incubated model halves. (C) n = 3, error bars show standard deviation. Significant differences to the water control in a Student’s t-test are marked by asterisks (p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***).

Figure 5

Ethanol as a solvent promotes the antimycotic activity of allicin via the gas phase. For gas phase experiments, synthetic allicin was diluted in either deionised water (diH₂O) or 96% ethanol. The lung model was coated the same as for the aerosol treatment (20 mL 2% (w/v) PDA with 10⁶ spores per mL of Paecilomyces formosus per half). The supply air was adjusted to 6 L per min, treatment duration was 20 min. The model was subsequently incubated at 37 °C. (A) After 24 h and 48 h of incubation, fungal growth was detected by staining with MTT. The lower half of the experiment model is shown. (B) Binary images of the 48 h incubated model halves. (C) n = 3, error bars show standard deviation. Significant differences between the treatments in a Student’s t-test are marked by asterisks (p < 0.001 = ***; n = non-significant).