Preliminary Study on Citrus Oils Antibacterial Activity Measured by Flow Cytometry: A Step-by-Step Development

Area of Science:

• Microbiology
• Natural Products Chemistry
• Analytical Chemistry

Background:

• Essential oils from Citrus sinensis (sweet orange) and Citrus latifolia (lime) possess known antiseptic, anti-inflammatory, and antioxidant properties.
• Evaluating their antibacterial capacity, alone or with antibiotics, offers potential for novel infection treatments.
• Flow cytometry provides a rapid and precise method for assessing antibacterial activity.

Purpose of the Study:

• To evaluate the antibacterial activity of Citrus essential oils against E. coli using flow cytometry.
• To investigate the synergistic or antagonistic effects of essential oil-antibiotic combinations.
• To propose standardization steps for flow cytometry-based antibacterial assays.

Main Methods:

• Utilized flow cytometry with SYTO9/PI staining to differentiate live and dead E. coli ATCC 25922 cells.
• Compared individual fluorochrome staining with dual staining, identifying issues with wavelength overlap in the latter.
• Established amikacin as a suitable positive control due to its dose-response behavior, unlike chloramphenicol and cefotaxime.

Main Results:

• Individual fluorochrome staining correlated well with viable cell counts, offering reliable results.
• Citrus essential oils exhibited low antibacterial activity (max 10-20%) compared to amikacin.
• Combinations of essential oils with amikacin demonstrated an unexpected and significant inhibition of the antibiotic's activity.

Conclusions:

• Standardized flow cytometry using individual fluorochrome staining is a viable method for measuring antibacterial activity.
• Citrus essential oils possess minimal direct antibacterial effects on E. coli.
• Essential oil-antibiotic combinations require careful investigation due to potential antagonistic interactions.