Efficacy of true cinnamon (Cinnamomum verum) leaf essential oil as a therapeutic alternative for Candida biofilm infections.

OBJECTIVES: The essential oil (EO) extracted from Cinnamomum verum leaves has been used as an antimicrobial agent for centuries. But its antifungal and antibiofilm efficacy is still not clearly studied. The objective of this research was to evaluate the in vitro antifungal and antibiofilm efficacy of C. verum leaf EO against C. albicans, C. tropicalis, and C. dubliniensis and the toxicity of EO using an in vitro model. MATERIALS AND METHODS: The effect of EO vapor was evaluated using a microatmosphere technique. CLSI microdilution assay was employed in determining the Minimum Inhibitory (MIC) and Fungicidal Concentrations (MFC). Killing time was determined using a standard protocol. The effect of EO on established biofilms was quantified and visualized using XTT and Scanning Electron Microscopy (SEM), respectively. Post-exposure intracellular changes were visualized using Transmission Electron Microscopy (TEM). The toxicological assessment was carried out with the Human Keratinocyte cell line. The chemical composition of EO was evaluated using Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: All test strains were susceptible to cinnamon oil vapor. EO exhibited MIC value 1.0 mg/ml and MFC value 2.0 mg/ml against test strains. The killing time of cinnamon oil was 6 hr. Minimum Biofilm Inhibitory Concentration (MBIC50) for established biofilms was <0.2 mg/ml for all test strains. SEM images exhibited cell wall damages, cellular shrinkages, and decreased hyphal formation of Candida. TEM indicated intracellular vacuolation, granulation, and cell wall damages. Cinnamon leaf oil caused no inhibition of HaCaT cells at any concentration tested. Eugenol was the abundant compound in cinnamon oil. CONCLUSION: C. verum EO is a potential alternative anti-Candida agent with minimal toxicity on the human host.

In-vitro Antibacterial and Antibiofilm Activity of Cinnamomum verum Leaf Oil against Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae.

Phytomedicines are becoming more popular in treatment of infectious diseases worldwide. Cinnamomum verum essential oil (EO) has been used as a therapeutic alternative for various diseases. This study aimed to evaluate the antibacterial and antibiofilm activity of the C. verum leaf EO against Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Effect of EO vapor on planktonic cells was determined using microatmosphere technique. CLSI M7-A10 method was employed in Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) experiments. Effect of EO on established biofilms was quantified and visualized using XTT and Scanning Electron Microscope (SEM). In-vitro toxicity was evaluated using Human Keratinocytes (HaCaT). Chemical analysis of EO was done using Gas Chromatography- Mass Spectrometry (GC-MS). All tested strains were sensitive to cinnamon oil vapor. EO exhibited 0.5 and 1.0 mg/mL MIC and MBC against all test strains. Minimum Biofilm Inhibitory and Biofilm Eradication Concentrations (MBIC50 and MBEC) were 1.0 and 4.0 mg/mL. SEM indicated cellular shrinkages, cell wall damages, and decreased biofilm densities. Cinnamon oil didn't show any toxicity on HaCaT cell at any concentration tested. Eugenol was the most abundant compound in C. verum oil. C. verum EO shows an antibacterial and antibiofilm activity with minimal toxicity on host.

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

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.

Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development

As there are sparse data on the impact of growth media on the phenomenon of biofilm development for Candida we evaluated the efficacy of three culture media on growth, adhesion and biofilm formation of two pathogenic yeasts, Candida albicans and Candida tropicalis. The planktonic phase yeast growth, either as monocultures or mixed cultures, in sabouraud dextrose broth (SDB), yeast nitrogen base (YNB), and RPMI 1640 was compared, and adhesion as well as biofilm formation were monitored using MTT and crystal violet (CV) assays and scanning electron microscopy. Planktonic cells of C. albicans, C. tropicalis and their 1:1 co-culture showed maximal growth in SDB. C. albicans/C. tropicalis adhesion was significantly facilitated in RPMI 1640 although the YNB elicited the maximum growth for C. tropicalis. Similarly, the biofilm growth was uniformly higher for both species in RPMI 1640, and C. tropicalis was the slower biofilm former in all three media. Scanning electron microscopy images tended to confirm the results of MTT and CV assay. Taken together, our data indicate that researchers should pay heed to the choice of laboratory culture media when comparing relative planktonic/biofilm growth of Candida. There is also a need for standardisation of biofilm development media so as to facilitate cross comparisons between laboratories.

Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development.

As there are sparse data on the impact of growth media on the phenomenon of biofilm development for Candida we evaluated the efficacy of three culture media on growth, adhesion and biofilm formation of two pathogenic yeasts, Candida albicans and Candida tropicalis. The planktonic phase yeast growth, either as monocultures or mixed cultures, in sabouraud dextrose broth (SDB), yeast nitrogen base (YNB), and RPMI 1640 was compared, and adhesion as well as biofilm formation were monitored using MTT and crystal violet (CV) assays and scanning electron microscopy. Planktonic cells of C. albicans, C. tropicalis and their 1:1 co-culture showed maximal growth in SDB. C. albicans/C. tropicalis adhesion was significantly facilitated in RPMI 1640 although the YNB elicited the maximum growth for C. tropicalis. Similarly, the biofilm growth was uniformly higher for both species in RPMI 1640, and C. tropicalis was the slower biofilm former in all three media. Scanning electron microscopy images tended to confirm the results of MTT and CV assay. Taken together, our data indicate that researchers should pay heed to the choice of laboratory culture media when comparing relative planktonic/biofilm growth of Candida. There is also a need for standardisation of biofilm development media so as to facilitate cross comparisons between laboratories.