Synergistic effects of carvacrol, α-terpinene, γ-terpinene, ρ-cymene and linalool against Gardnerella species

Abstract

Bacterial vaginosis (BV) is the most common vaginal infection affecting women worldwide. This infection is characterized by the loss of the dominant Lactobacillus community in the vaginal microbiota and an increase of anaerobic bacteria, that leads to the formation of a polymicrobial biofilm, mostly composed of Gardnerella spp. Treatment of BV is normally performed using broad-spectrum antibiotics, such as metronidazole and clindamycin. However, the high levels of recurrence of infection after treatment cessation have led to a demand for new therapeutic alternatives. Thymbra capitata essential oils (EOs) are known to have a wide spectrum of biological properties, including antibacterial activity. Thus, herein, we characterized two EOs of T. capitata and tested their antimicrobial activity as well as some of their main components, aiming to assess possible synergistic effects. Our findings showed that carvacrol and ρ-cymene established a strong synergistic antimicrobial effect against planktonic cultures of Gardnerella spp. On biofilm, carvacrol and linalool at sub-MIC concentrations proved more efficient in eliminating biofilm cells, while showing no cytotoxicity observed in a reconstituted human vaginal epithelium. The antibiofilm potential of the EOs and compounds was highlighted by the fact cells were not able to recover culturability after exposure to fresh medium.

Six Bacterial Vaginosis-Associated Species Can Form an In Vitro and Ex Vivo Polymicrobial Biofilm That Is Susceptible to Thymbra capitata Essential Oil

“…To better assess the EO antimicrobial potential against a BV polymicrobial biofilm, we also used an ex vivo model, wherein a polymicrobial biofilm was established on a reconstructed human vaginal epithelium. When applying a lower (0.32 µL mL -1 ), but non-cytotoxic concentration of the EO ( Sousa et al., 2022 ), a significant reduction of the biofilm biomass was also observed ( Figure 2A ), similar to what occurred in the in vitro experiment ( Figure 1A ). However, contrary to the in vitro experiment, EO exposure did not completely eradicate culturable cells, with only a 4-log reduction observed ( Figure 2B ).…”

“…It is characterized by a high percentage of oxygen-containing monoterpenes, being mainly composed of carvacrol (73.9%) followed by its biogenetic precursors, γ-terpinene (7.4%), and p-cymene (4.9%). The full composition of this EO has been described before ( Sousa et al., 2022 ). In vitro antibacterial activity of this EO against each species tested in the current study was evaluated by determining both MIC and MLC values.…”

“…We have previously shown T. capitata EO to be highly effective against Gardnerella spp. single-species biofilms and non-toxic for several lactobacilli ( Machado et al., 2017 ) while also not being toxic for the reconstructed human vaginal epithelium (at the used concentrations) ( Sousa et al., 2022 ). In the present study, the EO showed high antimicrobial activity at MLC concentration against the single-species biofilms.…”

“…The oil was isolated by hydrodistillation for 3 h from air-dried material, using a Clevenger -type apparatus according to the European Pharmacopoeia ( Council of Europe, 2010 ). EO composition was established by the combination of gas chromatography with FID detectors (GC-FID) and gas chromatography-mass spectroscopy (GC/MS) analysis, as previously reported ( Sousa et al., 2022 ). The EO components were identified by considering, concurrently: (i) the acquired retention indices on two columns with different phases, SPB-1 (polydimethylsiloxane) and SupelcoWax-10 (polyethyleneglycol), determined by linear interpolation relative to the retention times of C8–C23 of n-alkanes and compared with reference data from authentic products (available in the laboratory database of the Faculty of Pharmacy, University of Coimbra) and literature data ( Wallace, 2021 ); (ii) the acquired mass spectra compared with reference data from the laboratory database, the Wiley/NIST library ( McLafferty, 2009 ) and literature ( Adams, 2007 ).…”

Antioxidant and Anti-Inflammatory Effects of Thyme (Thymus vulgaris L.) Essential Oils Prepared at Different Plant Phenophases on Pseudomonas aeruginosa LPS-Activated THP-1 Macrophages

“…The main compound of TEO is thymol that possesses antibacterial, antiviral, antifungal, and anti-inflammatory properties [ 2 , 5 , 6 ]. Additional major components of TEO, such as carvacrol, p -cymene, γ -terpinene, and α -terpinene, also have antimicrobial, antioxidant, and anticancer activities [ 33 , 34 , 35 , 36 ].…”