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Daniela Machado1, Carlos Gaspar2,3, Ana Palmeira-de-Oliveira2,3, Carlos Cavaleiro4, Lígia Salgueiro4, José Martinez-de-Oliveira2,5 and Nuno Cerca1

1 Centre of Biological Engineering (CEB), Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), University of Minho, Campus de Gualtar, Braga, Portugal

2 CICS-UBI, Health Sciences Research Center, Faculty of Health Sciences, University of Beira Interior, Avenida Infante D. Henrique, Covilhã, Portugal

3 Labfit – HPRD: Health Products Research and Development Lda, Edificio UBIMEDICAL, Estrada Municipal 506, Covilhã, Portugal

4 CNC.IBILI, Faculty of Pharmacy, University of Coimbra, Azinhaga de S. Comba, Coimbra, Portugal 5 Women and Child Health Department, Centro Hospitalar Cova da Beira EPE, Covilhã, Portugal

*Correspondence: Nuno Cerca, Centre of Biological Engineering (CEB), Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Tel.: +351 253 60443, fax: +351 253 678 986; e-mail: nunocerca@ceb.uminho.pt Keywords: Bacterial vaginosis; Gardnerella vaginalis; lactobacilli; Thymbra capitata essential oil; carvacrol; biofilms. Abstract Aims: To evaluate the antibacterial activity of Thymbra capitata essential oil and its main compound, carvacrol, against Gardnerella vaginalis grown planktonically and as biofilms, and its effect of vaginal lactobacilli. Materials & methods: MIC, MLC and flow cytometry analysis were used to assess the antibacterial effect against planktonic cells. Anti-biofilm activity was measured through quantification of biomass and visualisation of biofilm structure by CLSM. Results: T. capitata essential oil and carvacrol exhibited a potent antibacterial activity against G. vaginalis cells. Anti-biofilm activity was more evident with the essential oil than carvacrol. Furthermore, vaginal lactobacilli were significantly more tolerant to the essential oil. Conclusion: T. capitata essential oil stands up as a promising therapeutic agent against G. vaginalis biofilm-related infections.

Introduction Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women in childbearing age and it has been related to serious health consequences, such as spontaneous abortion, preterm birth and postoperative infections [1]. Moreover, this condition is characterized by the replacement of lactobacilli by a significantly number of bacteria, mainly anaerobic, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., and Prevotella spp. [2,3]. Due to its polymicrobial nature, the etiology of BV is still poorly understood [4]. However, it is well established that BV involves the presence of a dense, structured, polymicrobial biofilm, predominantly composed of G. vaginalis clusters [5]. The role of the species less frequently found in BV in its aetiology is still a matter of debate, but a few studies clearly demonstrated a lower virulence potentials, as compared to G. vaginalis [6,7]. Nevertheless, it has been demonstrated that some secondary BV species might enhance G. vaginalis biofilm growth [8,9]. So, the current paradigm is that the establishment of G. vaginalis biofilm is an event required for the development and progression of BV [10].

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Importantly, standard antibiotics are often ineffective against BV, and this has been related with both high rates of bacterial resistance [11] and of resulting recurrence episodes [12]. In the attempt to overcome the high bacterial resistance and recurrence, natural compounds have been proposed as an emergent and valuable approach to treat BV [13]. Natural products, such as essential oils (EOs), have been used since ancient times due to their medicinal properties [14]. EOs are complex mixtures of volatile compounds, mainly monoterpenes, sesquiterpenes and phenylpropanoids, produced by aromatic plants [15]. Moreover, the use of EOs in human infections has been associated to a low risk of development of antimicrobial resistance [16], which constitutes a great advantage comparatively to antibiotics. Thymbra capitata (L.) Cav. [= Thymus capitatus Hoffms.et Link., Thymus creticus Brot., Corydothymus capitatus Rechenb. f., Satureja capitata L.] is an aromatic plant belonging to Lamiaceae family, widespread in the southern of Portugal [17]. This plant has been used for many commercial purposes, from culinary to cosmetic industries [18,19]. In Portuguese folk medicine, the EO extracted from this plant is used externally to treat oropharynx problems and in topical formulations to combat cutaneous infections, such as acne [20]. The study of T. capitata EO chemical composition revealed a high content in carvacrol [21] and previous in vitro studies have demonstrated that this EO displays a high antimicrobial activity against Listeria monocytogenes [22], Candida spp. [21], Aspergillus spp. and dermatophytes [23]. Due to its great potential as antimicrobial agent against a wide panoply of pathogens, we chose to test the potential of this EO against G. vaginalis, the main bacterial species involved in BV biofilms [5,10,24]. Thus, the main goal of the present study was to evaluate the antibacterial activity of T. capitata EO against G. vaginalis grown planktonically and as biofilm and to compare anti-G. vaginalis activity of EO with its major compound (purified carvacrol). Secondarily, we assessed the effect of T. capitata EO upon vaginal and probiotic lactobacilli species, in order, to assess the potential of T. capitata EO to disrupt the vaginal normal flora. Material & methods Plant material and EO extraction Aerial parts of T. capitata plant were collected at flowering stage from Algarve region (south of Portugal). Voucher specimens were deposited at the herbarium of the Department of Life Sciences of the University of Coimbra with the number LS430. T. capitata EO was obtained by water distillation for 3 h from air-dried material, using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia [25]. After extraction, the T. capitata EO were preserved in a sealed vial at 4ºC. The qualitative and quantitative analytical composition of the T. capitata EO was determined by gas chromatography and gas chromatography/mass spectrometry, which revealed that oil is mainly constituted by carvacrol (75.0%), y-terpinene (5.1%) and p-cymene (5.0%) [21]. Purified carvacrol (purchased from Sigma, St Louis, Missouri, USA) was used to determine the antimicrobial effect of the EO main component. Bacterial strains and growth conditions G. vaginalis is a capnophilic bacterium that grows well at 35 to 37ºC in humidified atmosphere of air plus 5 to 10% carbon dioxide (CO2) or in candle flame extinction jar [26]. Seven clinical strains of G. vaginalis isolated from women diagnosed with BV and identified by 16S RNA sequencing [7] were used in this study. G. vaginalis strains were kept frozen in Brain Heart Infusion Broth (BHI, Liofilchem, Roseto degli Abruzzi, Italy) with 23% (v/v) glycerol (Panreac, Castellar del Vallès, Barcelona, Spain) at -80ºC, until testing. For each experiment, G. vaginalis strains were grown on Columbia Blood Agar (CBA, Liofilchem) supplemented with 5% (v/v) of defibrinated horse blood (Oxoid Ltd, Basingstoke, Hampshire, United Kingdom) and incubated at 37ºC for 48 - 72 h under anaerobic conditions, in approximately 10% carbon dioxide (CO2), achieved by use of an incubator (Shel Lab, Cornelius, Oregon, USA). Furthermore, eight reference vaginal or probiotic lactobacilli strains namely, Lactobacillus acidophilus (KS400), Lactobacillus casei (ATCC393), Lactobacillus crispatus (KS116.1), Lactobacillus delbrueckii (DSM20081), Lactobacillus gasseri (KS114.1), Lactobacillus jensenii (KS119.1), Lactobacillus plantarum (ATCC8014) and Lactobacillus reuteri (DSM20016) were included in this work. For experiments involving lactobacilli, strains were subcultured in Man, Rogosa and Sharpe Agar (MRSA, VWR, Radnor, Pennsylvania, USA) and the agar plates were incubated at 37ºC for 48 h with 10% CO2 [27].

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Minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) determination MIC and MLC values of T. capitata EO in lactobacilli and G. vaginalis were determined by macrodilution broth method according to guidelines from Nature Protocols [28], with some minor modifications. Briefly, T. capitata EO were initially dissolved in dimethyl-sulfoxide (DMSO, Scharlau, Sentmenat, Spain). Then, serial dilutions of EO, ranging between 0.08 – 20 µL/mL, were prepared in sBHI [BHI supplemented with 2% (wt/v) gelatin (Liofilchem), 1% (wt/v) yeast extract (Liofilchem), 0.1% (wt/v) soluble starch (Panreac)] for experiments involving G. vaginalis strains, and in Man, Rogosa and Sharpe Broth (MRS, VWR, Radnor, Pennsylvania, USA) for experiments involving Lactobacillus strains. Bacterial suspensions were prepared by inoculating isolated colonies of the organisms (sBHI for G. vaginalis or MRS for lactobacilli), to a concentration of 106 CFU/mL, estimated using optical density (OD) at 600 nm (Bio-Tek Synergy HT, Winooski, Vermont, USA). The relationship between OD and cell concentration was determined previously by determining the concentration of the bacterial suspensions by plate count [29]. Afterwards, 500 µL of each dilution of EO was added into test tubes containing 500 µL of bacterial suspensions (106 CFU/mL). Also, MIC determination assays included a negative control, containing only medium, and positive control that contained bacterial suspension of lactobacilli or G. vaginalis adjusted to 106 CFU/mL and DMSO in the same concentration used to dilute the EO, in order to confirm that there was no interference of this organic solvent on bacterial growth. All test tubes were incubated for 48 h (lactobacilli) or 72 h (G. vaginalis) at 37ºC in a 10% CO2 atmosphere. After incubation, the MICs were evaluated by reading the turbidity, macroscopically, compared to a positive control. MIC value was defined as the lowest concentration of EO that inhibited visible bacterial growth (absence of turbidity). Moreover, MIC results were confirmed by measuring the OD at 600 nm of all test tubes. MLC were obtained following MIC determination by inoculating 10 µL of content of each test tube on MRSA (lactobacilli) or CBA (G. vaginalis) plates that incubated for 48 h (lactobacilli) or 72 h (G. vaginalis) at 37ºC in 10% CO2. MLC value was defined as the lowest concentration of EO that prevented the growth of treated bacteria on agar plates. All experiments were repeated three times on separate days with technical duplicates. Furthermore, the protocol described above was used to determine the antibacterial activity of carvacrol against G. vaginalis planktonic cells. Flow cytometry analysis The effect of the T. capitata EO and carvacrol on G. vaginalis planktonic cells was elucidated by flow cytometry, according with the protocol proposed by Pina-Vaz et al. [30], with some minor modifications. Briefly, after incubation of planktonic cells (108 CFU/mL) of strain UM066 for 1 h at 37°C in 10% CO2, the cells were stained for 15 min with propidium iodide, 1 µg/ml (PI, Sigma-Aldrich, St. Louis, Missouri, USA), protected from light, at room temperature. Following the staining step, the cells were analysed on the BD AccuriTM C6 (BD Accuri Cytometers Inc., Ann Arbor, Michigan, USA) flow cytometer at FL3 (620 nm red). Non-treated and non-PI-stained cells were used to determine the auto-fluorescence, while non-treated and stained cells were used as viability control, and bacterial cells treated with heat (70ºC) for 10 min were used as a dead cells control. For kinetic studies, bacterial cells were incubated during 5, 10, 15, 30 and 60 min with EO and carvacrol at MIC value and stained with PI using the protocol described above. Biofilm formation and quantification Biofilms were grown as previously described [31], with some minor modifications. Briefly, G. vaginalis cultures were adjusted to 108 CFU/mL in sBHI supplemented with 0.25% (wt/v) of glucose (sBHIG, Panreac AppliChem, Darmstadt, Germany). Then, 100 µL of each bacterial suspension of G. vaginalis isolates was dispensed into each well of 96-well microtiter plates (Orange Scientific, Braine-l'Alleud, Belgium). Afterwards, the microtiter plates were placed into an incubator at 37ºC under 10% CO2 during 48 h with replacement of consumed medium at 24 h of growth. After this time, EO (0.16; 0.32 and 0.64 µL/mL) and carvacrol (0.64 µL/mL – 4mM) were added and the microplates were incubated during 24 h at 37ºC with 10% CO2. Finally, biofilm biomass was quantified according the crystal violet (CV) staining method described by Peeters et al. [32]. In brief, after fixation step, biofilms were stained with 0.5% (wt/v) CV (Acros Organics, Morris Plains, New Jersey, USA). Next, biofilms were washed twice with phosphate buffered saline and bound CV was released with 33% (v/v) acetic acid (Fisher Scientific, Loughborough, Leicestershire, United Kingdom). Finally, OD was measured at 590 nm using the 96-well microplate reader (Bio-Tek Synergy HT, Winooski, Vermont, USA). All assays were repeated at least three times with eight technical replicates.

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Confocal laser scanning microscopy analysis (CLSM) of G. vaginalis biofilms Changes on the structure of G. vaginalis biofilms induced by T. capitata EO and carvacrol were evaluated by CLSM. For this experiment, strain UM121 was incubated on 8-well chamber slide (Thermo Fisher Scientific™ Nunc™ Lab-Tek™, Rochester, New York, USA) at 37ºC in 10% CO2 during 48 h with replacement of sBHIG medium at 24 h of growth. Afterwards, EO (0.64µL/mL) and carvacrol (0.64µL/mL) were added and the chamber slides were incubated during 24 h at 37ºC with 10% CO2. Then, used medium was removed and biofilms were stained with SYTO® BC (Molecular Probes Inc., Eugene, Oregon, USA). After staining, the biofilms were gently rinsed with 0.9% sodium chloride (Liofilchem). The CLSM images were acquired in an OlympusTM FluoView FV1000 (Olympus, Lisboa, Portugal) confocal scanning laser microscope, using a 10x objective. All assays were repeated three times on separate days. Statistical analysis Data were analysed using independent samples T-test with statistical package for the social science 17.0 software (SPSS; Chicago, Illinois, USA). Statistical differences in the effect of T. capitata EO and carvacrol on G. vaginalis biofilm biomass were considered significant at P values < 0.05. Results and discussion The low efficacy of antibiotics available to treat BV and high associated recurrence rates has led to the search for novel therapeutic strategies combining effectivity and low cost. Previous studies demonstrated that T. capitata EO [21–23] and its main compound (carvacrol) [33,34] display a wide spectrum of antimicrobial activity. In this sense, we decided to evaluate the antibacterial activity of this oil and of carvacrol against G. vaginalis, the major species contributing to the development of the BV biofilm [5,10,24]. We first determined the MIC and MLC of both EO and carvacrol using several clinically isolates of G. vaginalis. As shown in Table 1, T. capitata EO and carvacrol exhibited a potent antibacterial activity against all isolates tested, since they presented low values of MIC (0.16 µL/mL) and MLC (0.16 µL/mL). Interestingly, MIC was the same as MLC, suggesting a bactericidal effect of both the oil and carvacrol. Table 1 | Anti-Gardnerella vaginalis activity of Thymbra capitata essential oil and of carvacrol in planktonic cells.

Strains T. capitata essential oil (µL/mL) Carvacrol (µL/mL) MIC MLC MIC MLC

UM034 0.16 0.16 0.16 0.16 UM035 0.16 0.16 0.16 0.16 UM066 0.16 0.16 0.16 0.16 UM067 0.16 0.16 0.16 0.16 UM121 0.16 0.16 0.16 0.16 UM137 0.16 0.16 0.16 0.16 UM241 0.16 0.16 0.16 0.16

Note: Carvacrol concentration of 0.16 µL/mL is equivalent to 1 mM. Flow cytometry analysis was used to evaluate the effect of the EO and carvacrol on the integrity of G. vaginalis planktonic cells, using PI as fluorescent marker. PI is a nucleic acid stain that only penetrates cells that are dead or got severe cytoplasmatic membrane damage [35]. As shown in Figure 1A, both T. capitata EO and carvacrol presented a quick bactericidal effect against G. vaginalis after incubation during 1 h at MIC value, which is confirmed by the increase in PI staining. Furthermore, by evaluating the effect of T. capitata EO and carvacrol over short periods of time, it was possible to conclude that a high percentage of cell death was observable after only 5 min of incubation (Figure 1B). These results are in accordance with previous reports that demonstrated that T. capitata EO and carvacrol were able to induce primary lesion of the membrane with subsequent cell death in other microbial species [23,34].

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Fig. 1 | Flow cytometric analysis of Thymbra capitata essential oil and of carvacrol effect against Gardnerella vaginalis UM066 planktonic cells. A) Histogram representing Propidium iodide (PI)-stained cells. Viable = non-treated cells (viable control); dead = cells treated with heat at 70º C (dead control); T. capitata and Carvacrol (cells treated with T. capitata essential oil and with carvacrol at minimal inhibitory concentration (MIC) value during 60 min). B) Kinetic study showing the percentage (%) of PI-stained cells after treatment with T. capitata essential oil and carvacrol at MIC value during 5, 10, 15, 30 and 60 min.

Next, we evaluated the effect of T. capitata EO and carvacrol on G. vaginalis biofilm biomass (Figure 2). Strain to strain variability was detected in the inhibitory effect of EO and carvacrol. Even so, T. capitata EO at 0.64 µL/mL was effective against all biofilms tested, confirmed by higher percentages of biofilm biomass reduction (49-91%). In contrast, carvacrol in the same concentration (0.64 µL/mL) had a lower anti-biofilm effect. We further analyse the impact of T. capitata EO or carvacrol on G. vaginalis biofilms by CLSM. Interestingly, we verified that the T. capitata EO and carvacrol were both able to disrupt G. vaginalis biofilm, causing a cellular density reduction (Figure 3). However, the cell clusters present in the biofilms exposed to carvacrol were bigger than those found in biofilms exposed to T. capitata EO, suggesting that T. capitata EO had a greater ability to disrupt G. vaginalis biofilm structure than carvacrol. These results might be, however, less worrisome since it is has been shown that in vivo G. vaginalis biofilms are less thickly than they formed in vitro [24]. Independently of the differences between in vivo and in vitro biofilm formation, our results are promising and clearly demonstrate the potential of the EO and/or carvacrol as anti G. vaginalis agents.

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Fig. 2 | Effect of Thymbra capitata essential oil (EO) and of carvacrol upon Gardnerella vaginalis biofilm biomass. A) Biomass of biofilms determined by measurement of optical density (OD) at 590 nm of stained biofilms by crystal violet method. B) Percentage (%) of reduction of biomass of G. vaginalis biofilms exposed to T. capitata EO at 0.16, 0.32 and 0.64 µL/mL or to carvacrol at 0.64 µL/mL. Errors bars represent standard error of the mean. Statistical differences in the % of biofilm biomass reduction using T. capitata EO (0.64 µL/mL) or carvacrol (0.64 µL/mL) are marked with * (P <0.05).

Despite the in vitro antibacterial activity of T. capitata EO against G. vaginalis biofilms, a possible negative outcome could be its effect of the normal flora of the vagina, particularly the beneficial lactobacilli. In this sense, we evaluated the susceptibility of several Lactobacillus species to T. capitata EO. As can be seen in Table 2, both vaginal or common probiotic species of lactobacilli demonstrated a significantly higher tolerance to the T. capitata, showing MIC (1.25 - 2.50 µL/mL) and MLC values (1.25 - 2.50 µL/mL) near 10 fold higher than G. vaginalis. These findings highlight an added advantage of this EO, as compared to metronidazole, the most commonly used antibiotic against BV [36], since it suggest that a proper dosage would affect G. vaginalis without harming the beneficial resident lactobacilli. The fact that EO has a better anti-biofilm activity is not entirely surprising and is in accordance with previous studies that demonstrated that EOs had higher antimicrobial activity than their major compounds [37,38]. The advantage of using EO instead of the isolated compounds is supported by the knowledge that the effect of the EO is often greater than the sum of the effects of its isolated compounds, due to synergistic effects and complementary activities between major and minor components [37–39]. For this reason, many advocate that EO should be used a whole, instead of

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trying to isolate the major compounds. The ability of EOs disrupts microbial structures has been attributed to its high hydrophobicity, that causes a cascade of events, including degradation of the cell membrane, cytoplasm coagulation and leakage of the cell contents[40–42]. However it is important to note that some chemical variability has been observed when comparing different samples of T. capitata EOs [21], a common result of using natural products, but confirming problems related to plant extracts dosage and reproducibility. For this reason, the applicability of carvacrol alone would be of high interest. Alternatively, it would be very advantageous to monitor the crop conditions of T. capitata plant in order to promote the quality and chemical homogeneity of the EO, or to guarantee the composition through blending.

Fig. 3 | Effect of Thymbra capitata essential oil and of carvacrol on Gardnerella vaginalis UM121 biofilm structure. (A) 48 h biofilm without antimicrobial agents; (B) 72 h biofilm without antimicrobial agents; (C) Biofilm exposed to T. capitata essential oil at 0.64 µL/mL for 24 h; (D) Biofilm exposed to carvacrol at 0.64 µL/mL for 24 h.

Table 2 | Susceptibility of lactobacilli strains to Thymbra capitata essential oil

Strains T. capitata essential oil (µL/mL) MIC MLC

Lactobacillus acidophilus KS400b 2.50 2.50 Lactobacillus casei ATCC393b 2.50 2.50 Lactobacillus crispatus KS116.1a 2.50 2.50 Lactobacillus delbrueckii DSM20081b 1.25 1.25 Lactobacillus gasseri KS114.1a 2.50 2.50 Lactobacillus jensenii KS119.1a 1.25 1.25 Lactobacillus plantarum ATCC8014b 2.50 2.50 Lactobacillus reuteri DSM20016b 2.50 2.50

a corresponds to common vaginal species; b non-vaginal probiotic species.

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Conclusion In this study, we report for the first time the antibacterial activity of T. capitata EO and carvacrol upon G. vaginalis planktonic cultures and biofilms. Interestingly, both compounds demonstrated a potent antibacterial activity against G. vaginalis planktonic cells. However, a significant anti-biofilm activity was only evident with the EO, suggesting that anti-biofilm effect of the oil is not only dependent on the EO major component. Importantly, T. capitata EO had a significantly lower antibacterial effect against lactobacilli. Thus, T. capitata EO stands up as a promising therapeutic agent for G. vaginalis biofilm-related infections with high potential application as topical formulation. This principle was demonstrated by Palmeira-de-Oliveira et al. as they incorporated T. capitata EO in chitosan hydrogel [43]. The resulting TCCH hydrogel presented an acidic nature, compatible with the vaginal pH, being active against Candida planktonic cells and biofilms. Due to high antimicrobial activity, TCCH hydrogel was proposed as a putative therapeutic tool for the treatment of vulvovaginal candidosis [43]. Nevertheless, the present study presents some limitations, mainly related to the in vitro model used here. Indeed, it has been described that microbicides are less active in vivo than in vitro [44]. Therefore, further investigations are required toward determining whether T. capitata EO maintains this antimicrobial activity in vivo. A first approach would be to test this using an ex vivo vaginal mucosa model [45]. This would allow not only to determine possible cytotoxic effects but to determine if a similar anti-biofilm effect could be observed. Executive summary

• T. capitata EO and carvacrol display a potent antibacterial activity against G. vaginalis planktonic cells.

• Anti-biofilm activity of T. capitata EO is higher than carvacrol. • T. capitata EO is significantly less active against lactobacilli strains than G. vaginalis. • T. capitata EO is a promising alternative to treat bacterial vaginosis and other G. vaginalis

biofilms related infections. Financial & competing interests’ disclosure The authors are grateful to Foundation for Science and Technology (FCT, Portugal) for D. Machado grant (SFRH/BD/87569/2012) and financial support (FCT Strategic Project of UID/BIO/04469/2013 unit). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. References 1. Schwebke JR. New concepts in the etiology of bacterial vaginosis. Curr. Infect. Dis. Rep. 11(2),

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