Introduction: The aim of the study was the evaluation of the antimicrobial and antibiofilm properties of Cannabis sativa essential oil (EO) versus Staphylococcus aureus. Materials and Methods: The essential oil was obtained by hydrodistillation of fresh aerial parts from hemp variety C. sativa cv. Futura75 cultivated in Abruzzo region. The antimicrobial and antibiofilm activity of Hemp EO were evaluated on one reference strain Staphylococcus aureus (Sa) ATCC 29213 and three clinical strains of Sa 101, Sa 104, Sa 105, isolated by different sources and characterized for the antimicrobial susceptibility pattern. The determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) was carried out by using the broth microdilution method according to the NCCLS guidelines and the alamarBlue® (AB) viability assay. Hemp EO was prepared as 10% (v/v) solutions in ethanol and was used in the range of 0.5-16 mg/mL. The antibiofilm effect of Hemp EO was determined by the evaluation of Minimum Biofilm Eradication Concentration (MBEC) by using the broth microdilution method in the range of 16-32 mg/mL and the AB assay. The MBEC was confirmed by Live/Dead Cell Viability Staining and fluorescent microscopy analysis. However, Colony Forming Unit (CFU) enumeration was performed to evaluate the bacterial cell viability in the biofilm and planktonic phenotypes. Using a validated HPLC-PDA method was also obtained phenolics profile of the EO in order to correlate the biological activities with chemical profile on selected compounds. Results: The Hemp EO showed a MIC corresponding to 8 mg/mL versus all S. aureus strains including S. aureus 104, a multi-drug resistant strain isolated by a pharyngeal swab of a male patient. With regard to MBC the Hemp EO showed to be effective at 16 mg/mL versus all S. aureus strains. Furthermore, the Hemp EO showed its capability to eradicate a mature biofilm developed by S. aureus showing a MBEC at 24 mg/mL towards all the strains of S. aureus except for S. aureus 105 that showed a MBEC corresponding to 16 mg/mL. The effect of the Hemp EO in the eradication of S. aureus pre-formed biofilms were confirmed by the statistical significant reduction of the CFU count. The Live/Dead staining and fluorescent microscopy evaluation showed a well-structured biofilm constituted by a great amount of live cells. On the contrary, S. aureus mature biofilms treated with 24 mg/mL of Hemp EO, did not show a reduction of the biofilm biomass, however, the biofilms treated with Hemp EO were characterized by a multitude of dead cells. Discussion and Conclusions: The antibacterial and antibiofilm activities of Hemp EO suggest it a possible candidate in the treatment of the infections associated with S. aureus.

Antimicrobial and Antibiofilm Activities of the Italian Cannabis sativa L Essential Oil against Staphylococcus aureus

PUCA, VALENTINA;SIMONE CARRADORI;MARCELLO LOCATELLI;LUIGI MENGHINI;ROSSELLA GRANDE
2018

Abstract

Introduction: The aim of the study was the evaluation of the antimicrobial and antibiofilm properties of Cannabis sativa essential oil (EO) versus Staphylococcus aureus. Materials and Methods: The essential oil was obtained by hydrodistillation of fresh aerial parts from hemp variety C. sativa cv. Futura75 cultivated in Abruzzo region. The antimicrobial and antibiofilm activity of Hemp EO were evaluated on one reference strain Staphylococcus aureus (Sa) ATCC 29213 and three clinical strains of Sa 101, Sa 104, Sa 105, isolated by different sources and characterized for the antimicrobial susceptibility pattern. The determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) was carried out by using the broth microdilution method according to the NCCLS guidelines and the alamarBlue® (AB) viability assay. Hemp EO was prepared as 10% (v/v) solutions in ethanol and was used in the range of 0.5-16 mg/mL. The antibiofilm effect of Hemp EO was determined by the evaluation of Minimum Biofilm Eradication Concentration (MBEC) by using the broth microdilution method in the range of 16-32 mg/mL and the AB assay. The MBEC was confirmed by Live/Dead Cell Viability Staining and fluorescent microscopy analysis. However, Colony Forming Unit (CFU) enumeration was performed to evaluate the bacterial cell viability in the biofilm and planktonic phenotypes. Using a validated HPLC-PDA method was also obtained phenolics profile of the EO in order to correlate the biological activities with chemical profile on selected compounds. Results: The Hemp EO showed a MIC corresponding to 8 mg/mL versus all S. aureus strains including S. aureus 104, a multi-drug resistant strain isolated by a pharyngeal swab of a male patient. With regard to MBC the Hemp EO showed to be effective at 16 mg/mL versus all S. aureus strains. Furthermore, the Hemp EO showed its capability to eradicate a mature biofilm developed by S. aureus showing a MBEC at 24 mg/mL towards all the strains of S. aureus except for S. aureus 105 that showed a MBEC corresponding to 16 mg/mL. The effect of the Hemp EO in the eradication of S. aureus pre-formed biofilms were confirmed by the statistical significant reduction of the CFU count. The Live/Dead staining and fluorescent microscopy evaluation showed a well-structured biofilm constituted by a great amount of live cells. On the contrary, S. aureus mature biofilms treated with 24 mg/mL of Hemp EO, did not show a reduction of the biofilm biomass, however, the biofilms treated with Hemp EO were characterized by a multitude of dead cells. Discussion and Conclusions: The antibacterial and antibiofilm activities of Hemp EO suggest it a possible candidate in the treatment of the infections associated with S. aureus.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11564/696340
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