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

Abstract

Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.

Keywords: alternative therapy; bacterial vaginosis (BV); essential oils; microbial interactions; polymicrobial biofilms.

Figure 1

Single-species and polymicrobial BV biofilms. (A) Biomass quantification of the single-species and polymicrobial biofilms prior to and after EO exposure using crystal violet method. (B) Culturable cells quantification from the single-species and polymicrobial biofilms prior to and after EO treatment, by CFU method. (C) Determination of the percentage distribution of each bacterial species in 24 h polymicrobial BV biofilm, by qPCR. i.CT stands for initial control, before the medium replacement; f.CT stands for final control, after incubation with fresh medium. Statistically significant differences between polymicrobial vs single-species biofilms are represented with ^α for 24 h incubation time and with ^β for 48 h incubation time (two-way ANOVA and Dunnett’s multiple comparisons test, p < 0.05). Values are significantly different for ^* i.CT vs f.CT, ^τ i.CT vs EO, and ^γ f.CT vs EO (two-way ANOVA and Tukey’s multiple comparisons test, p < 0.05). Gardnerella vaginalis (Gv); Fannyhessea vaginae (Fv); Lactobacillus iners (Li); Mobiluncus curtisii (Mc); Peptostreptococcus anaerobius (Pa); Prevotella bivia (Pb); Essential oil (EO); Limit of detection (LOD).

Figure 2

Polymicrobial BV biofilm developed on a reconstructed human vaginal epithelium. (A) Periodic Acid-Schiff staining images with the BV biofilm before and after EO treatment at 600× magnification. (B) Cells culturability from the control and EO treated biofilms as determined by CFU method. (C) Bacterial distribution in the polymicrobial BV biofilms as determined by qPCR after extraction of genomic DNA of each bacterial species. ^*Values are significantly different for control vs EO-exposed biofilms (paired t-test, p < 0.05). Essential oil (EO); Limit of detection (LOD). More examples are shown in Supplementary Material.