Citrus bergamia: Kinetics of Antimicrobial Activity on Clinical Isolates

Abstract

Background: The inappropriate use of antibiotics has increased selective pressure and the spread of multi-drug-resistant (MDR) pathogens, which reduces the possibility of effective treatment. A potential alternative therapeutic approach may be represented by essential oils, such as the distilled extract of bergamot (Citrus bergamia Risso et Poiteau). Such natural products exercise numerous biological activities, including antimicrobial effects.

Methods: This work aimed to evaluate the kinetics of the bactericidal and fungicidal activity of the distilled extract of bergamot on MDR bacteria and fungi from clinical specimens using the time-kill assay. Furthermore, the antimicrobial activity of the distilled extract of bergamot on the morphology and cellular organization of clinical pathogens was evaluated by confocal laser scanning microscopy.

Results: Our results demonstrated that the distilled extract of bergamot exhibited significant antimicrobial activity and a specific bactericidal effect against the bacterial and fungal strains tested. Furthermore, confocal microscope images clearly showed compromised membrane integrity, damage and cell death in bacterial samples treated with the distilled extract of bergamot. In addition, progressive alterations in cell-wall composition, cytoplasmic material and nucleus structure triggered by exposure to the distilled extract of bergamot were identified in the fungal samples considered.

Conclusions: Our data suggest that the use of essential oils, such as distilled extract of bergamot (Citrus bergamia Risso et Poiteau), can represent a valid alternative therapeutic strategy to counteract antibiotic resistance of pathogens.

Keywords: Citrus bergamia; antimicrobial activity; fungicidal activity; multi-drug-resistant bacteria; time-kill assay.

Figure 1

Citrus bergamia Risso et Poiteau.

Figure 2

Time-kill curves of distilled extract of bergamot 1 × MBC against: (A) Staphylococcus aureus strains; (B) Staphylococcus coagulase-negative strains; (C) Acinetobacter baumannii complex strains; (D) Klebsiella pneumoniae strains; (E) Candida albicans strains; (F) Candida spp. strains.

Figure 3

Representative images of confocal laser scanning microscopy of MDR bacteria treated with the distilled extract of bergamot and stained with acridine orange. “Untreated”: control of bacterial growth; “treated”: strains treated with the distilled extract of bergamot (MBC). Green fluorescence shows the presence of living cells; red fluorescence shows the presence of dead cells.

Figure 4

Confocal laser scanning microscopy of cell wall components of C. albicans 1/18 clinical isolate treated with the distilled extract of bergamot for 60′. Double-staining of chitin and mannan fluorescently labeled with Calcofluor white (CFW) and Concanavalin A (Conc A), respectively, was performed. “Untreated”: growth control of C. albicans 1/18; “treated”: C. albicans 1/18 treated with the distilled extract of bergamot (MBC). The reduction in fluorescence intensity shows a decrease in the relative cell wall components.

Figure 5

Confocal laser scanning microscopy of C. albicans 1/18 clinical isolate exposed for up to 60′ to the distilled extract of bergamot. DAPI staining for DNA identification was performed. “Untreated”: growth control of C. albicans 1/18; “treated”: C. albicans 1/18 treated with the distilled extract of bergamot.