GC-MS analysis of Myrtus communis extract and its antibacterial activity against Gram-positive bacteria

Abstract

Background: Myrtus communis is a typical plant of Mediterranean area. The different parts of this plant such as berries, branches, and leaves have been used worldwide as a traditional/folk medicine for the treatment of various ailments and diseases.

Methods: Ethanolic leaf extract of the plant was prepared by Soxhlet extraction method. Zone of inhibition, minimum inhibitory concentration and minimal bactericidal concentration were determined by well diffusion method and microplate alamar blue assay. GC-MS analysis was carried out to identify the compounds present in the extract. Microscopy and ImageJ software were used respectively for morphology and cell-length measurements. GraphPad Prism was used for statistical analysis.

Results: The ethanolic extract showed strong inhibitory effect against Gram-positive and acid-fast bacteria with significant inhibition-zone size (9-25 mm), MIC (4.87-78 μg/ml), as well as MBC (0.3-20 mg/ml). However, no effect was observed on the growth of Gram-negative bacteria. The growth inhibition was found to be associated with the damage of cell wall as the extract-treated cells were sensitive to cell wall-targeting antibiotics and displayed the cell wall damage-depicting morphological defects. GC-MS analysis confirmed the presence of novel compounds in addition to the most representative compounds of the essential oils/extracts of M. communis of other country origins.

Conclusion: These results demonstrate that M. communis leaf extract could be the source of compounds to be used for the treatment of Gram-positive bacterial infections. This is the first report, which provides insights into the mechanism of action of the extract in inhibiting the growth of Gram-positive bacteria.

Keywords: Cell wall; GC-MS; Gram-negative bacteria; Gram-positive bacteria; Myrtus communis; Plant extract.

Fig. 1

Sensitivity of extract-treated M. smegmatis and S. aureus cells to antibiotics. Cells after treatment with ethanolic extract were washed and re-suspended in nutrient broth containing 1x alamar blue. The cultures were aliquoted in triplicate wells of a microplate containing an antibiotic (colistin, tetracycline or levofloxacin). For the control, no antibiotic but water was added. Absorbance at 570 nm was measured at regular time intervals of 3 hours (M. smegmatis) or 30 min (S. aureus). For each antibiotic, the average values of A₅₇₀ (YY-axis) of the extract-treated M. smegmatis (a) or S. aureus (b) culture were plotted over the time (XX-axis). Error bars; ± SD

Fig. 2

Morphology of the extract-treated S. aureus and M. smegmatis cells. Cells of S. aureus and M. smegmatis were individually treated with ethanolic leaf extract of M. communis. For the control, cells were treated with ethanol only. Both the control and extract treated cells were observed under the microscope using the 100x objective. (a & b) S. aureus cells treated with ethanol and extract, respectively. (c & d) M. smegmatis cells treated with ethanol and extract, respectively. (e) Distribution of M. smegmatis cells based on their length after treating separately with ethanol (untreated) and leaf extract (treated). Scale bar; 1 μm