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. 2023 Aug 30;9(9):888.
doi: 10.3390/jof9090888.

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain

Carolina Zapata-Zapata  1 , Mauricio Rojas-López  2   3 , Liliana T García  4 , Wendy Quintero  4 , María C Terrón  5 , Daniel Luque  5 , Ana C Mesa-Arango  1
Affiliations

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain

Carolina Zapata-Zapata et al. J Fungi (Basel). .

Abstract

Candida tropicalis is one of the most pathogenic species within the genus. Increased antifungal resistance has been reported, which is in part due to the organism's ability to form biofilms. In natural products derived from plants, such as essential oils (EOs) or their major components, there is significant potential to develop new antifungals or to both enhance the efficacy and reduce the toxicity of conventional antifungals. This study aimed to evaluate the effect of combining an EO of Lippia origanoides or thymol with fluconazole on an azole-resistant C. tropicalis strain. Synergism was observed in the combination of fluconazole with the EO and with thymol, and minimal inhibitory concentrations for fluconazole decreased at least 32-fold. As a consequence of the synergistic interactions, mitochondrial membrane potential was reduced, and mitochondrial superoxide production increased. Alteration in nuclear morphology, cell surface, and ultrastructure was also observed. In conclusion, the synergistic interaction between L. origanoides EO or thymol with fluconazole reverted the azole-resistant C. tropicalis phenotype. These findings suggest that L. origanoides EO or thymol alone, or in combination with fluconazole, have the potential for development as antifungal therapies for this yeast, including resistant strains.

Keywords: Candida tropicalis; Lippia origanoides; antifungal activity; essential oil; synergism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Isobologram of EO/FLC (A) and thymol/FLC (B) interaction against C. tropicalis strains. Points below the additivity line indicate a synergistic effect.
Figure 2
Figure 2
Effect of the different treatments on C. tropicalis cell mitochondrial membrane potential (∆ψm) and plasmatic membrane. (A,C) Photos of cells stained with JC-1 obtained by fluorescence microscopy. Green indicates monomers, and yellow indicates aggregates. (B,D) Representative plots of percentages of DiOC6(3) uptake cells (high (H) and low (L)). PI-positive cell percentages are shown above. Scale bar: 25 µm.
Figure 3
Figure 3
Candida tropicalis ATCC 200956 cells stained with MitoSOXTM Red were observed under a fluorescence microscope. (A1) Untreated cells; (A2) cells treated with EO, (A3) thymol, (A4) EO/FLC, (A5) thymol/FLC, and (A6) FLC. (B) Mean fluorescence intensity values of cells with different treatments. ** p = 0.0008; *** p < 0.0001. Scale bar: 25 µm.
Figure 4
Figure 4
Fluorescence microscopy pictures depicting the changes on the nucleus of C. tropicalis ATCC 200956. (A) Untreated and treated cells with (B) EO, (C) thymol, (D) EO/FLC, (E) thymol/FLC, and (F) FLC. Scale bar: 25 µm.
Figure 5
Figure 5
Effect of different treatments on the C. tropicalis ATCC 200956 cell cycle. (A) Representative plots of data indicating 1 MFI (red), 1.5 MFI (green), and 2 MFI (blue) cell percentage. (B) Average 1.5 MFI + 2 MFI cell percentage. MFI: Median fluorescence intensity. p-values: EO = 0.0056; EO/FLC = 0.0028; thymol/FLC = 0.0016; FLC = 0.0007. (** p < 0.01; ***p < 0.001).
Figure 6
Figure 6
SEM images of C. tropicalis ATCC 200956: (A) untreated cells; (B) treated with EO, (C) thymol, (D) EO/FLC, (E) thymol/FLC, (F) FLC, (G) AMB, and (H) CSF. Arrows indicate alterations on the cell surface. AMB: amphotericin B; FLC: fluconazole; CSF: caspofungin; EO: essential oil.
Figure 7
Figure 7
TEM micrographs of C. tropicalis ATCC 200956. (A) Untreated cells and cells treated with (B) EO, (C) thymol, (D) EO/FLC, (E) thymol/FLC, and (F) FLC. PM: plasmatic membrane; CW: cell wall; M: mitochondria; N: nucleus. Scale bar: 500 nm.
Figure 8
Figure 8
Candida tropicalis ATCC 200956 cell wall thickness measurement. **** p < 0.0001.
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