Effect of Cinnamomum verum leaf essential oil on virulence factors of Candida species and determination of the in-vivo toxicity with Galleria mellonella model

Abstract

Background: Essential oils (EO) extracted from Cinnamomum verum has been used as an antimicrobial agents for centuries. The effects of C. verum leaf oil against virulence of microorganisms is not well studied yet.

Objectives: This study evaluates the effect of C. verum leaf oil against three virulence factors of Candida albicans, C. tropicalis and C. dubliniensis and its in-vivo toxicity.

Methods: Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing Candida cells to adhere to the polystyrene surface for 2 h. Biofilm formation of Candida in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. In-vivo toxicity of EO was determined using Galleria mellonella larvae. Chlorhexidine digluconate: positive control.

Results: Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by C. albicans, C. tropicalis and C. dubliniensis with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC50 for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of Candida. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with in-vivo experiment model at any concentration tested.

Conclusion: C. verum leaf oil acts against virulence factors of Candida and does not show any toxicity.

Fig. 1:. percentage reduction in XTT metabolic activity of Candida albicans (ATCC MYA-2876), C. tropicalis (ATCC 750) and C. dubliniensis (ATCC MYA-646) in the presence of different concentrations of Cinnamomum verum (CV) leaf oil and chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD).

Fig. 2:. percentage germ tube formation of (A) Candida albicans (ATCC MYA-2876) and (B) C. dubliniensis (ATCC MYA-646) with 0.25, 0.5 and 1.0 mg/mL Cinnamomum verum leaf oil and chlorhexidine digluconate within 6 h test period with 2 h intervals. 0 mg/mL concentration indicates the negative control. All error bars represent the ± 2 standard deviations (SD).

Fig. 3:. percentage reduction in XTT metabolic activity of Candida albicans (ATCC MYA-2876), C. tropicalis (ATCC 750) and C. dubliniensis (ATCC MYA-646) forming biofilms with the presence of different concentrations of Cinnamomum verum (CV) leaf oil and chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD). All error bars represent the ± 2 standard deviations (SD).

Fig. 4:. Log colony forming unit (CFU) values of forming Candida albicans (CA) (ATCC MYA-2876), C. tropicalis (CT) (ATCC 750) and C. dubliniensis (CD) (ATCC MYA-646) biofilms in the presence of different concentrations of (A) Cinnamomum verum (CV) leaf oil and (B) chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD).

Fig. 5:. scanning electron microscopy (SEM) images of Candida albicans (ATCC MYA-2876) forming biofilms in the presence of 0.5 mg/mL Cinnamomum verum leaf oil (A and D), 1.0 mg/mL C. verum leaf oil (B and E), 2 mg/mL C. verum leaf oil (C and F), 0.25 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K). I and L: negative control. Red circles: cell wall deformities with treatments.

Fig. 6:. scanning electron microscopy (SEM) images of Candida tropicalis (ATCC 750) forming biofilms in the presence of 0.5 mg/mL Cinnamomum verum leaf oil (A and D), 1.0 mg/mL C. verum leaf oil (B and E), 2.0 mg/mL C. verum leaf oil (C and F), 0.125 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K) I and L: negative control. Red circle: cell wall deformities with treatments. Red solid arrow: leakages of intracellular components.

Fig. 7:. scanning electron microscopy (SEM) images of Candida dubliniensis (ATCC MYA-646) forming biofilms in the presence of 0.5 mg/mL Cinnamomum verum leaf oil (A and D), 1.0 mg/mL C. verum leaf oil (B and E), 2mg/mL C. verum leaf oil (C and F), 0.125 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K) I and L: negative control. Red circle: cell wall deformities with treatments.

Fig. 8:. (A) Time Lapses images of developing biofilms of Candida albicans (ATCC MYA-2876). Each column represents the concentration of Cinnamomum verum leaf oil treatment. (B) Time Lapses images of developing biofilms of C. tropicalis (ATCC 750). Each column represents the concentration of C. verum leaf oil treatment. (C) Time Lapses images of developing biofilms of C. dubliniensis (ATCC MYA-646). Each column represents the concentration of C. verum leaf oil treatment.

Fig. 9:. survival rate of Galleria mellonella larvae after administration of Cinnamomum verum leaf oil over five days experiment period. Control curve was obtained by administrating sterile phosphate buffered saline (PBS) in to larvae.