Abstract
Essential oils of Citrus sinensis and Citrus latifolia have shown biological functions as antiseptics, anti-inflammatories, antioxidants, antifungal and antimutagenic, so the evaluation of their antibacterial capacity, by themselves or in combination with standard antibiotics, presents an alternative for infection treatment. Flow cytometry opens the door for the design of faster and more accurate measurement of antibacterial activity. We use a SYTO9/PI staining system on E. coli ATCC 25922 to determine antibacterial activity by counting live and dead cells through flow cytometry. We found that dual staining showed highly variable results due to wavelength overlapping and instead we used fluorochrome individual staining that highly correlated with viable counts. Chloramphenicol and cefotaxime treatments did not present a dose-response behavior, rendered diffuse readings and/or gave filament formation on fluorescence microscopy. Amikacin was a better comparison standard because it presented a dose-response behavior. Essential oils had low antibacterial activity as compared to amikacin, with a maximum of 10% and 20% for C. latifolia and C. sinensis, respectively. Combinations of essential oils with antibiotic resulted in an unforeseen strong inhibition of amikacin activity. Although a low antibacterial activity was found, a series of standardization steps are proposed for antibacterial activity measurement by flow cytometry.
Highlights
The World Health Organization has identified antibacterial resistance as one of the most dangerous threats to human health worldwide
In here we aimed to evaluate the accuracy of SYTO9/Propidium Iodide (PI) dual staining in flow cytometry (FC) measurement of the possible antibacterial activity of Citrus latifolia and Citrus sinensis essential oils, simultaneously comparing the results to viable count method, with the purpose of establishing if FC could be a plausible test for the determination of bioactivity of these and other substances as alternatives or adjuvants of antibiotics
Populations with low numbers of dead cells had a marked displacement from the expected selecting gates (Figure 1b) even reaching the region of live bacteria; to avoid confusing readings, 70% isopropanol was preferred as dead cells control
Summary
The World Health Organization has identified antibacterial resistance as one of the most dangerous threats to human health worldwide. The increasing number of resistant pathogens has compromised the efficacy of antibiotic stewardship and rates of associated morbimortality and economic burden have become seriously elevated in recent times [1] This phenomenon has motivated the development of new antibacterial alternatives for both prophylactic and therapeutic purposes and the research of those with a natural origin. EOs are highly soluble in lipids and organic compounds and are generally liquid at room temperature, some could be solid or resinous [4,5]. They are a mixture of monoterpenes, sesquiterpenes, oxygenated derivatives, low weight alkanes, alcohols, aldehydes, ketones, esters and acids, associated with non-volatile paraffins and waxes [6]. EOs are present in all plant organs, including buds, flowers, leaves, seeds, stems, fruits, roots, wood and bark, there is a general purveyance within secretory cells, cavities, channels and cells of the epidermis [7]
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