Studies Regarding the Antimicrobial Behavior of Clotrimazole and Limonene

Abstract

The paper presents the results of the studies performed to establish the effect of the mixtures between limonene and clotrimazole against microbial pathogens involved in dermatological diseases, such as Candida albicans, Staphyloccocus aureus, and Escherichia coli. Preliminary data obtained from the studies performed in microplates revealed a possible synergism between the mixture of clotrimazole and limonene for Staphylococcus aureus. Studies performed "in silico" with programs such as CLC Drug Discovery Workbench and MOLEGRO Virtual Docker, gave favorable scores for docking each compound on a specific binding site for each microorganism. The tests performed for validation, with the clotrimazole (0.1%) and different sources of limonene (1.9% citrus essential oils), showed a synergistic effect on Staphylococcus aureus in the case of the mixtures between clotrimazole and the essential oils of Citrus reticulata or Citrus paradisi. The studies performed on Staphylococcus aureus MRSA showed a synergistic effect between clotrimazole and the essential oils obtained from Citrus bergamia, Citrus aurantium, or Citrus paradisi. In the case of Pseudomonas aeruginosa, essential oils and clotrimazole used alone did not exhibit antimicrobial activities, but the mixtures between clotrimazole and the essential oils of Citrus bergamia or Citrus sinensis exhibited a synergistic antimicrobial effect.

Keywords: clotrimazole; limonene; synergistic effect.

Figure 1

The methodology presented in studies regarding the antimicrobial behavior of clotrimazole and limonene.

Figure 2

Influence of limonene exposure on S. aureus growth, after 24 h. At a concentration of limonene in the culture medium higher than 2500 μg/mL, S. aureus is inhibited.

Figure 3

Influence of CT exposure on S. aureus growth, after 24 h. At a concentration of clotrimazole in the culture medium higher than 156 μg/mL, S. aureus is inhibited.

Figure 4

Influence of (limonene + CT) exposure on S. aureus growth, after 24 h. At a concentration of clotrimazole in the culture medium higher than 312 μg/mL, S. aureus is inhibited.

Figure 5

Influence of limonene exposure on C. albicans growth, after 48 h. At a concentration of limonene in the culture medium higher than 1250 μg/mL, C. albicans is inhibited.

Figure 6

Influence of CT exposure on C. albicans growth, after 48 h. At a concentration of clotrimazole in the culture medium higher than 312 μg/mL, C. albicans is inhibited.

Figure 7

Influence of (CT + limonene) exposure on C. albicans growth, after 48 h. At a concentration of limonene + clotrimazole in the culture medium higher than 1250 μg/mL, C. albicans is inhibited.

Figure 8

Molecular docking of the co-crystallized (NDP), limonene, and clotrimazole for C. albicans in the binding site 1AI9. (a1) Co-crystallized NDP (score: −79.35; RMSD 2.86) in the binding site (model generated by CLC); (b1) the co-crystallized NDP (score: −155.24; RMSD 0.00) in the binding site (model generated with MOLEGRO); (a2) hydrogen bonds of the co-crystallized NDP in the binding site, generated by CLC; (b2) hydrogen bonds, electrostatic interactions, steric interactions between the co-crystallized NDP and amino acids in 3D, generated by MOLEGRO; (a3) the interaction of co-crystallized NDP with amino acids’ residues in binding site, generated by CLC; (b3) hydrogen bonds (blue), electrostatic interactions (green), steric interactions (red) between the co-crystallized NDP and amino acids in 2D, generated by MOLEGRO; (a4) the interaction of clotrimazole with the amino acid residues from the binding site (model generated by CLC); (b4) the interactions of clotrimazole and the hydrogen bonds (blue line) with THR 58 amino acid in the binding site (model generated by MOLEGRO); (a5) the interaction of limonene with the amino acid residues from the binding site in a model generated with CLC; (b5) the interaction of limonene with the amino acid residues from the binding the site (model generated by MOLEGRO).

Figure 9

Molecular docking of co-crystallized (TOP), limonene, and clotrimazole for S. aureus in binding site 2W9H. (a1) Docking of the co-crystallized TOP (score: −53.12; RMSD 0.35) in the binding site of 2W9H (model generated by CLC); (b1) docking of the co-crystallized TOP (score: −100.32; RMSD 0.00) in the binding site of 2W9H (model generated by MOLEGRO); (a2) interactions between the co-crystallized TOP and the amino acid residues in the binding site 2W9H (model generated by CLC). The blue lines represent the hydrogen bonds; (b2) interactions between the co-crystallized TOP and the amino acid residues in the binding site of 2W9H (model generated with MOLEGRO). The blue lines represent the hydrogen bonds; (a3) hydrogen bonds (blue dotted lines) between clotrimazole and SER 49 amino acid in the binding site 2W9H, generated by CLC; (b3) hydrogen bonds (blue) and steric interactions (red) between clotrimazole and amino acids in the binding site 2W9H in the 2D format, generated by MOLEGRO; (a4) the interactions between the clotrimazole and the amino acid residues in the binding site 2W9H, generated by CLC; (b4) hydrogen bonds and steric interactions, between clotrimazole and the amino acids, in the binding site 2W9H, generated by MOLEGRO; (a5) interactions of limonene with the amino acid residues in the binding site of 2W9H, generated by CLC; (b5) interactions of limonene with the amino acid residues in the binding site 2W9H, generated by MOLEGRO.

Figure 10

Molecular docking of the co-crystallized 8XQ, limonene, and clotrimazole for E. coli in the binding site 4JHT. (a1) Docking of the co-crystallized 8XQ in the binding site 4JHT generated by CLC); (b1) docking of the co-crystallized in the binding site 4JHT, generated by MOLEGRO); (a2) docking of the co-crystallized 8XQ, hydrogen bonds, and interactions with amino acid residues from the binding site 4JHT, generated by CLC; (b2) hydrogen bonds, electrostatic interactions, and steric interactions between the co-crystallized 8XQ and the amino acids from the binding site 4JHT, in 3D model generated by MOLEGRO; (a3) interactions between clotrimazole and the amino acid residues from the binding site 84JHT, generated by CLC; (b3) hydrogen bonds and steric interactions between clotrimazole and the amino acids from the binding site 4JHT, generated with MOLEGRO; (a4) the hydrogen bonds (blue dotted lines) between clotrimazole and SER 49, in the binding site 4JHT, generated by CLC; (b4) the hydrogen bonds (blue) and steric interactions (red) between clotrimazole and the amino acids from the binding site 4JHT, 2D model generated by MOLEGRO; (a5) the interactions of limonene with the amino acid residues from the binding site 4JHT, generated by CLC; (b5) the interactions of limonene with the amino acid residues from the binding site 4JHT, generated by MOLEGRO.

Figure 11

Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w), on C. albicans. No synergism between clotrimazole and the different sources of limonene was observed.

Figure 12

Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w), on E.coli. No synergism between clotrimazole and the different sources of limonene was observed.

Figure 13

Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w) on S. aureus. The synergistic effect is evident in the case of the mixtures between clotrimazole and the essential oils of mandarin (red mandarin or green mandarin), and, respectively, between clotrimazole and the essential oil of grapefruit.

Figure 14

Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w), on S. aureus MRSA. The synergistic effect is evident in the case of the mixtures between clotrimazole and the essential oils of bergamot (two sources of this essential oil).

Figure 15

Influence of different sources of limonene (citrus essential oil, 1.9%, w/w) and of the mixture of them with clotrimazole (0.1%, w/w) on P. aeruginosa. The synergistic effect is evident in the case of the mixtures between clotrimazole and essential oils of Citrus bergamia and Citrus sinensis.