Extra Virgin Olive Oil-Based Formulations: A “Green” Strategy against Chlamydia trachomatis

In recent decades, antibiotic misuse has emerged as an important risk factor for the appearance of multi-drug-resistant bacteria, and, recently, antimicrobial resistance has also been described in Chlamydia trachomatis as the leading cause of bacterial sexually transmitted diseases worldwide. Herein, we investigated, for the first time, the antibacterial activity against C. trachomatis of a polyphenolic extract of extra virgin olive oil (EVOO), alongside purified oleocanthal and oleacein, two of its main components, in natural deep eutectic solvent (NaDES), a biocompatible solvent. The anti-chlamydial activity of olive-oil polyphenols (OOPs) was tested in the different phases of chlamydial developmental cycle by using an in vitro infection model. Transmission and scanning electron microscopy analysis were performed for investigating potential alterations of adhesion and invasion, as well as morphology, of chlamydial elementary bodies (EBs) to host cells. The main result of our study is the anti-bacterial activity of OOPs towards C. trachomatis EBs down to a total polyphenol concentration of 1.7 μg/mL, as shown by a statistically significant decrease (93.53%) of the total number of chlamydial-inclusion-forming units (p < 0.0001). Transmission and scanning electron microscopy analysis supported its anti-chlamydial effect, suggesting that OOP might damage the chlamydial outer layers, impairing their structural integrity and hindering EB capability to infect the host cell. In conclusion, OOPs may represent an interesting alternative therapeutic option toward C. trachomatis, although further studies are necessary for exploring its clinical applications.


Introduction
Chlamydia trachomatis is an obligate intracellular pathogen with a peculiar biphasic developmental cycle occurring into a phagocytic vacuole termed inclusion [1]. It alternates between two different forms, the extracellular infectious elementary body (EB), responsible for adhesion and invasion into the host cell, and the intracellular replicative reticulate body (RB), responsible for multiplication within the inclusion [2]. The entire duration of the C. trachomatis developmental cycle ranges approximately between 36 and 48 h, ending with the release of chlamydial EBs from infected cells and their dissemination within the host [3].
C. trachomatis is the leading cause of bacterial sexually transmitted diseases worldwide, with more than 130 million new cases per year according to the most recent World Health Organization (WHO) estimates [4]. The primary manifestations of C. trachomatis genital infection are urethritis, cervicitis, and salpingitis in women, although the majority of infections are asymptomatic [5,6]. Indeed, approximately 80% of women with oleacein and oleocanthal are present in large amounts compared to the other polyphenols. In particular, the concentrations of hydroxytyrosol, tyrosol, oleacein, and oleocanthal in OOP were 0.39 ± 0.06 mg/mL, 0.99 ± 0.17 mg/mL, 4.35 ± 0.21 mg/mL, and 9.61 ± 0.62 mg/mL, respectively (Supplementary Figure S1). Furthermore, the total polyphenol content, measured using the Folin-Ciocalteu assay and expressed as hydroxytyrosol equivalents, is 16.95 ± 2.22 mg/mL. The total polyphenol content and the single-compound concentrations of OOP are reported in Table 1. The values are reported as mean ± standard deviation.

Cytotoxicity of OOP, Oleacein and Oleocanthal
Preliminary investigations were carried out to determine the effect of OOP, oleacein and oleocanthal, dissolved in NaDES, on the viability of McCoy cells, in order to verify that these substances did not exert a direct cytotoxic effect on cells and to optimize the amount of mixture to be used in anti-chlamydial experiments.
As shown in Figure 1, a statistically significant decrease in McCoy cell viability was evidenced for OOP, oleacein and oleocanthal in NaDES starting from a 1:200 dilution ratio (corresponding to a polyphenol concentration of 85 µg/mL for OOP, and a concentration of 0.25 mM for oleacein and oleocanthal). Interestingly, oleacein showed a lower cytotoxicity on McCoy cells as compared to oleocanthal at the concentrations of 0.5 mM (32.03% decrease in cell viability vs. 84.00%, respectively, p < 0.0000001) and 0.25 mM (16.51% decrease in cell viability vs. 71.78%, respectively, p < 0.00001). hydroxytyrosol, tyrosol, oleacein and oleocanthal, similar to previous preparations [23]. Quantitative analysis of the chromatographic peaks showed that oleacein and oleocanthal are present in large amounts compared to the other polyphenols. In particular, the concentrations of hydroxytyrosol, tyrosol, oleacein, and oleocanthal in OOP were 0.39 ± 0.06 mg/mL, 0.99 ± 0.17 mg/mL, 4.35 ± 0.21 mg/mL, and 9.61 ± 0.62 mg/mL, respectively (Supplementary Figure S1). Furthermore, the total polyphenol content, measured using the Folin-Ciocalteu assay and expressed as hydroxytyrosol equivalents, is 16.95 ± 2.22 mg/mL. The total polyphenol content and the single-compound concentrations of OOP are reported in Table 1.

Cytotoxicity of OOP, Oleacein and Oleocanthal
Preliminary investigations were carried out to determine the effect of OOP, oleacein and oleocanthal, dissolved in NaDES, on the viability of McCoy cells, in order to verify that these substances did not exert a direct cytotoxic effect on cells and to optimize the amount of mixture to be used in anti-chlamydial experiments.
As shown in Figure 1, a statistically significant decrease in McCoy cell viability was evidenced for OOP, oleacein and oleocanthal in NaDES starting from a 1:200 dilution ratio (corresponding to a polyphenol concentration of 85 µg/mL for OOP, and a concentration of 0.25 mM for oleacein and oleocanthal). Interestingly, oleacein showed a lower cytotoxicity on McCoy cells as compared to oleocanthal at the concentrations of 0.5 mM (32.03% decrease in cell viability vs. 84.00%, respectively, p < 0.0000001) and 0.25 mM (16.51% decrease in cell viability vs. 71.78%, respectively, p < 0.00001).
NaDES alone also showed a statistically significant cytotoxicity on McCoy cells up to the dilution ratio 1:200 (15.6% decrease in cell viability), albeit with a lower reduction of cell viability than that observed in the presence of OOP (58%), oleacein (16.5%) and oleocanthal (71.8%), as shown in Supplementary Figure S2.

Anti-C. trachomatis Activities of OOP, Oleocanthal and Oleacein
Initially, the anti-bacterial properties of NaDES alone against C. trachomatis were investigated in the same experimental conditions as the EVOO-based formulations; no statistically significant decrease in the total number of chlamydial IFUs was observed at either the pre-treatment, pre-incubation or treatment phases ( Figure S3).
Then, the anti-chlamydial activity of OOP, oleacein and oleocanthal was evaluated directly against extracellular C. trachomatis EBs (pre-treatment phase), at the concentrations reported in Table 2. A significant reduction of chlamydial infectious EBs was observed for OOP at all the concentrations investigated (p < 0.05, Figure 2 and Table 2), as well as for oleocanthal and oleacein in NaDES (p < 0.01 and p < 0.05, respectively, Figure 3 and Table 2), as compared to untreated C. trachomatis EBs. Furthermore, the anti-chlamydial activity showed a dose-dependent effect, as evidenced by a decrease in chlamydial inhibition at lower concentrations ( Table 2). NaDES alone also showed a statistically significant cytotoxicity on McCoy cells up to the dilution ratio 1:200 (15.6% decrease in cell viability), albeit with a lower reduction of cell viability than that observed in the presence of OOP (58%), oleacein (16.5%) and oleocanthal (71.8%), as shown in Supplementary Figure S2.

Anti-C. trachomatis Activities of OOP, Oleocanthal and Oleacein
Initially, the anti-bacterial properties of NaDES alone against C. trachomatis were investigated in the same experimental conditions as the EVOO-based formulations; no statistically significant decrease in the total number of chlamydial IFUs was observed at either the pre-treatment, pre-incubation or treatment phases ( Figure S3).
Then, the anti-chlamydial activity of OOP, oleacein and oleocanthal was evaluated directly against extracellular C. trachomatis EBs (pre-treatment phase), at the concentrations reported in Table 2. A significant reduction of chlamydial infectious EBs was observed for OOP at all the concentrations investigated (p < 0.05, Figure 2 and Table 2), as well as for oleocanthal and oleacein in NaDES (p < 0.01 and p < 0.05, respectively, Figure 3 and Table 2), as compared to untreated C. trachomatis EBs. Furthermore, the anti-chlamydial activity showed a dose-dependent effect, as evidenced by a decrease in chlamydial inhibition at lower concentrations ( Table 2).     Lastly, we evaluated the anti-chlamydial activity of OOP, oleacein and oleocanthal in NaDES by treating McCoy cells before (pre-incubation phase) and after (treatment phase) C. trachomatis infection at non-cytotoxic concentrations: 17 µg/mL and 1.7 µg/mL for OOP, and 0.05 mM and 0.005 mM for both oleacein and oleocanthal. As shown in Figure 4, no statistically significant decrease in the total number of C. trachomatis IFUs was observed in the pre-incubation and treatment phases for OOP, oleacein and oleocanthal in NaDES at all the concentrations tested. Lastly, we evaluated the anti-chlamydial activity of OOP, oleacein and oleo in NaDES by treating McCoy cells before (pre-incubation phase) and after (tr phase) C. trachomatis infection at non-cytotoxic concentrations: 17 µg/mL and 1.7 for OOP, and 0.05 mM and 0.005 mM for both oleacein and oleocanthal. As show ure 4, no statistically significant decrease in the total number of C. trachomatis IF observed in the pre-incubation and treatment phases for OOP, oleacein and oleo in NaDES at all the concentrations tested.

Transmission Electron Microscopy of C. trachomatis EB Adhesion and Invasion
Given the high anti-chlamydial activity observed when C. trachomatis EBs w treated with OOP in NaDES, at a concentration of 170 µg/mL, we aimed to inv whether this effect could alter chlamydial adhesion and invasion of host-cell mem and, hence, the development of nascent inclusions via transmission electron micro As shown in Figure 5, untreated C. trachomatis EBs are able to adhere to the ce brane, followed by their internalization via endocytosis, as evidenced by the pres chlamydial EBs into the host-cell cytoplasm. On the contrary, the treatment of C matis EBs with OOP renders them unable to adhere to and invade the host cell, as

Transmission Electron Microscopy of C. trachomatis EB Adhesion and Invasion
Given the high anti-chlamydial activity observed when C. trachomatis EBs were pretreated with OOP in NaDES, at a concentration of 170 µg/mL, we aimed to investigate whether this effect could alter chlamydial adhesion and invasion of host-cell membranes and, hence, the development of nascent inclusions via transmission electron microscopy.
As shown in Figure 5, untreated C. trachomatis EBs are able to adhere to the cell membrane, followed by their internalization via endocytosis, as evidenced by the presence of chlamydial EBs into the host-cell cytoplasm. On the contrary, the treatment of C. trachomatis

Scanning Electron Microscopy of OOP-Treated C. trachomatis EBs
Given the absence of chlamydial EB adhesion and invasion within host cells following pre-treatment with OOP at the concentration of 170 µg/mL, as evidenced by transmission electron microscopy, we aimed to investigate their potential ultrastructural changes via scanning electron microscopy.
As shown in Figure 6, untreated C. trachomatis EBs appear as clusters of spherical particles with a diameter of approximately 200 to 300 nm, consistent with the typical C. trachomatis EB size as reported in the literature [25]. Conversely, scanning electron micrographs of C. trachomatis EBs treated with 170 µg/mL of OOP show the presence of a network of aberrant spherical particles with a much smaller diameter (5-10 nm), indicating the absence of morphologically normal chlamydial EBs.

Scanning Electron Microscopy of OOP-Treated C. trachomatis EBs
Given the absence of chlamydial EB adhesion and invasion within host cells following pre-treatment with OOP at the concentration of 170 µg/mL, as evidenced by transmission electron microscopy, we aimed to investigate their potential ultrastructural changes via scanning electron microscopy.
As shown in Figure 6, untreated C. trachomatis EBs appear as clusters of spherical particles with a diameter of approximately 200 to 300 nm, consistent with the typical C. trachomatis EB size as reported in the literature [25]. Conversely, scanning electron micrographs of C. trachomatis EBs treated with 170 µg/mL of OOP show the presence of a network of aberrant spherical particles with a much smaller diameter (5-10 nm), indicating the absence of morphologically normal chlamydial EBs.
4, x FOR PEER REVIEW 8 of 15 Figure 6. Scanning electron micrographs of C. trachomatis EBs treated with OOP. C. trachomatis EBs were pre-treated with OOP in NaDES (170 µg/mL). Then, chlamydial EBs were examined via scanning electron microscopy, at different magnification levels (2 µm, 1 µm and 0.5 µm). Yellow arrows point to the spherical particles whose size is compatible with that of morphologically normal C. trachomatis EBs.

Discussion
Herein, for the first time, the anti-microbial activity of different green OOP-based Figure 6. Scanning electron micrographs of C. trachomatis EBs treated with OOP. C. trachomatis EBs were pre-treated with OOP in NaDES (170 µg/mL). Then, chlamydial EBs were examined via scanning electron microscopy, at different magnification levels (2 µm, 1 µm and 0.5 µm). Yellow arrows point to the spherical particles whose size is compatible with that of morphologically normal C. trachomatis EBs.

Discussion
Herein, for the first time, the anti-microbial activity of different green OOP-based formulations in NaDES towards C. trachomatis has been investigated. The main result of our study is compelling evidence that OOP possessed a potent anti-chlamydial activity down to a total non-cytotoxic polyphenol concentration of 1.7 µg/mL, with a dose-dependent effect. Similarly, its purified active compounds, oleacein and oleocanthal, also showed a significant anti-chlamydial activity. In particular, OOP-based formulations were effective towards C. trachomatis EBs, the extracellular forms responsible for transmission of the infection and dissemination into the host, limiting, hence, the development of chronic complications. Interestingly, OOP was characterized by the highest anti-chlamydial activity, as evidenced by a >90% reduction of C. trachomatis IFUs. The anti-microbial effect against chlamydial EBs was confirmed by transmission electron microscopy analysis, revealing the absence of chlamydial EBs attached to cell membranes or internalized in phagocytic vacuoles after OOP treatment. Scanning electron microscopy also supported anti-chlamydial EB activity, highlighting chlamydial extracellular forms with a consistent decrease in their size following treatment with OOP, suggesting that it might damage the chlamydial outer layers, impairing their structural integrity.
The discovery of novel "green" OOP-based formulations as anti-chlamydial agents is highly relevant in light of the fact that, in recent years, the problem of antimicrobial resistance has also acquired growing importance in C. trachomatis, as suggested by significant treatment failure rates following first-line antimicrobials like doxycycline or azithromycin [26]. In particular, treatment failure ranged from 5% to 23% depending upon the population examined, and further evidence showed higher treatment failure rates (up to 14%) during azithromycin treatment than with doxycycline [9,27,28]. These observations are not surprising since in vitro studies have reported that azithromycin resistance could be raised through mutations in C. trachomatis 23S rRNA genes [10,29]. Moreover, other genetic mutations responsible for resistance to different microbial drugs were described, including, for example, tet(M) gene for tetracycline resistance [10,28,30]. Consequently, the phenomenon of C. trachomatis antibiotic resistance is highly likely, although, to date, no chlamydial resistant strains have been isolated; this may be explained, indeed, by the relatively low sensitivity of culture methods for the isolation of C. trachomatis (up to 50%), especially when compared to modern nucleic acid amplification tests [31,32].
More importantly, there are increasing concerns that tetracycline resistance, found in C. suis isolates from pigs and then associated with the resistance gene tetC [33,34], can be transferred into C. trachomatis strains. Indeed, gene transfer between C. suis and C. trachomatis can occur in nature, as evidenced by the observation of 16s rRNA genes from C. suis, in different C. trachomatis isolates, and by in vitro studies showing horizontal transfer of tetR from C. suis to human clinical isolates of C. trachomatis following coculture [35,36].
Interestingly, in our study, OOP showed higher anti-chlamydial activity as compared to its main active components alone, oleacein and oleocanthal, and this may be attributed to the extraction method using NaDES, a mix of natural compounds (betaine:propylene glycol), that has led to an increased yield of secoiridoids. As a matter of fact, the main advantage of using polyphenols from EVOO in NaDES compared to single purified polyphenols is the lower production cost of the extract compared to individual compounds, making it accessible for in vivo experiments or human use. Furthermore, the extraction of polyphenols from EVOO using NaDES presents significant advantages over the use of traditional organic solvents typically employed for the preparation of these extracts. NaDES are biocompatible "green" solvents that pose no risks to humans or the environment, unlike, for example, organic solvents such as methanol. Moreover, the preparation time of the extracts is considerably reduced, since in a single step, it is possible to obtain a concentrated extract of polyphenols without the need to remove the organic solvent [37]. This is, indeed, an efficient preparative method that allows one to obtain concentrated extracts with an easy, rapid, and eco-friendly procedure, that can be applied to many other plants, food matrices or food wastes for the extraction of bioactive molecules.
Limitations of this study include the lack of synergism tests with the antibiotics recommended for treating chlamydial genital infections, like azithromycin or doxycycline, as well as drug-resistant strains of C. trachomatis.
In conclusion, the promising anti-chlamydial properties of OOP, alongside the biocompatible and eco-friendly extraction method with a low impact on human health and the environment, suggest this formulation as a potential alternative therapeutic option for C. trachomatis genital infections, although further studies are necessary for exploring its clinical applications.

Preparation and Analysis of the Antimicrobial Agents
Polyphenolic extract was prepared from extra virgin olive oil (EVOO) obtained by cold pressing of the drupes of Olea europaea L. (Coratina cultivar), purchased from a local market in the Puglia region (Corato, Italy). The polyphenolic fraction was extracted as described in [37]. The NaDES was prepared by mixing two components (betaine:propylene glycol 1:3.5 molar ratio) at 70 • C under magnetic stirring for 1 h. After cooling, EVOO was added to the NaDESs in a 1:50 v/v (NaDES:EVOO) ratio. The extraction was carried out under magnetic stirring at room temperature for 15 min and then transferred via a separatory funnel for decantation and phase separation. The extract was analysed to determine the total polyphenol content and polyphenolic composition. Total phenols were determined by the Folin-Ciocalteu assay [38] by mixing 790 µL of distilled water with 10 µL of standard, sample, or blank. To these, 50 µL of Folin-Ciocalteu reagent (Merck KGaA, Darmstadt, Germany) was added, incubated for 3 min at room temperature, and then 150 µL of 20% (w/v) Na 2 CO 3 was added. After 2 h of incubation, the absorbance at 760 nm was measured using a Hitachi U2000 spectrophotometer (Hitachi, Tokyo, Japan). The results were expressed as hydroxytyrosol equivalents. The extracts were also assayed by UHPLC-DAD/MS, by utilizing a Waters Acquity H-Class UPLC system, as previously described by Francioso et al. (2020) [37]. The chromatographic system was coupled to a photodiode array (DAD PDA) and a single-quadruple mass detector with an electrospray ionization source (QDa). Chromatography was performed on a reverse-phase C18 column (Phenomenex Kinetex, 100 mm × 2.1 mm i.d., 2.6 µm particle size). Solvent A was 0.1% aqueous formic acid (Merck), and solvent B was 0.1% formic acid in methanol (UPLC gradient grade, Merck). The flow rate was 0.5 mL/min, and the column temperature was set at 35 • C. Elution was performed with a linear gradient from 2 to 100% B in a total time of 17 min including re-equilibration. The samples were diluted in the mobile phase and injected through the needle. The photodiode array detector was set up in the range of 200-600 nm. Mass spectrometric detection was performed in the negative electrospray ionization mode, using nitrogen as the nebulizer gas. Analyses were performed in the total ion current (TIC) mode with a mass range of 50-1000 m/z. The capillary voltage was 0.8 kV, cone voltage 15 V, ion source temperature 120 • C, and probe temperature 600 • C. Compounds were identified by retention time, m/z, UV-VIS spectrum, and comparing them with commercially available standards (Merck). Quantification of each compound was performed by using standard calibration curves in the range of 0.1-2 nmol. Oleacein and oleocanthal single molecules, were provided by Active-Italia S.r.l. freeze-dried and dissolved in NaDES at final concentration of 50 mM (16.0 mg/mL and 15.2 mg/mL for oleacein and oleocanthal, respectively).

Cell Culture and Culture Conditions
The McCoy cell line (ECACC, Public Health England, catalogue number 90010305, Porton Down, Salisbury, UK) was seeded in 25 cm 2 cell culture flasks with ventilated caps and grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 • C in a humidified atmosphere with 5% CO 2 . Upon confluency (>85%), cells were passaged with brief trypsinization.

Propagation and Titration of C. trachomatis
C. trachomatis serovar D strain UW3 (VR-855, ATCC, Manassa, VA, USA) was propagated in McCoy cells, as previously described [1]. Briefly, confluent McCoy cell monolayers were infected with chlamydial EBs by centrifugation at 750× g for 30 min, and then harvested by scraping after 36 to 40 h post infection. The resulting suspension was, then, vortexed with sterile glass beads for 2 min and, after the removal of cell debris, the supernatant, containing chlamydial EBs, was added to an equal volume of 4× Sucrose Phosphate (4SP) buffer, and stored at −80 • C.
C. trachomatis titration was performed via immunofluorescence assay (IFA); briefly, Mc-Coy cell monolayers were infected with 10-fold serial dilutions of bacterial stock, incubated for 48 h at 37 • C in humidified atmosphere with 5% CO 2 , fixed with methanol and stained with isothiocyanate-conjugated monoclonal antibody anti-C. trachomatis LPS (Merifluor ® Chlamydia, Meridian Bioscience Inc., Cincinnati, OH, USA), as previously described [39]. The total number of C. trachomatis inclusion-forming units (IFUs) was enumerated by counting all microscope fields using a fluorescence microscope (400× magnification).

Effects of OOP, Oleocanthal and Oleacein on the Different Phases of C. trachomatis Developmental Cycle
OOP, oleocanthal and oleacein in NaDES, as well as NaDES alone, were tested at increasing concentrations for their anti-chlamydial activity on the different phases of the C. trachomatis developmental cycle, specifically the pre-treatment of chlamydial EBs, the pre-incubation of McCoy cell monolayers followed by C. trachomatis EB infection, and the treatment of McCoy cell monolayers infected with C. trachomatis EBs.

Pre-Treatment Phase
To detect the activity of OOP, oleocanthal and oleacein in NaDES, as well as NaDES alone, on C. trachomatis serovar D, 5000 EBs/mL, corresponding to a multiplicity of infection (MOI) of 0.05, were pre-incubated in DMEM supplemented with 10% FBS, in the absence or presence of OOP, oleocanthal and oleacein in NaDES for 1 h at 37 • C in humidified atmosphere with 5% CO 2 . Subsequently, C. trachomatis EB suspension, containing the tested compounds, was further diluted 10 times in DMEM supplemented with 10% FBS, and, hence, used to infect McCoy cell monolayers grown on glass coverslips in 24-well cell-culture trays by centrifugation at 750× g for 30 min at room temperature. Subsequently, the cells were washed with DPBS to remove non-internalized C. trachomatis EBs and newly incubated in fresh culture medium, consisting of DMEM supplemented with 10% FBS. After 36 h of incubation at 37 • C in a humidified atmosphere with 5% CO 2 , the total number of C. trachomatis IFU was determined by IFA.

Pre-Incubation Phase
McCoy cell monolayers, grown on glass coverslips in 24-well cell-culture trays, were pre-incubated in DMEM supplemented with 10% FBS, in the absence or presence of OOP, oleocanthal and oleacein in NaDES, as well NaDES alone. After 24 h of incubation at 37 • C in a humidified atmosphere with 5% CO 2 , the cell culture medium containing the tested compounds was removed by washing the cells 3 times with DPBS, and then, McCoy cell monolayers were infected with C. trachomatis at a MOI of 0.05 as described above. After 36 h of incubation at 37 • C in humidified atmosphere with 5% CO 2 , the total number of C. trachomatis IFU was determined by IFA.

Treatment Phase
McCoy cell monolayers, grown on glass coverslips in 24-well cell-culture trays, were infected with C. trachomatis at an MOI of 0.05, by centrifugation at 750× g for 30 min at room temperature. Subsequently, the cells were washed with DPBS to remove non-internalized C. trachomatis EBs and fresh medium, with or without OOP, oleocanthal and oleacein in NaDES, as well as NaDES alone, was added to the infected cells. After 36 h of incubation at 37 • C in humidified atmosphere with 5% CO 2 , the total number of C. trachomatis IFU was determined by IFA.

Scanning Electron Microscopy of Pre-Treated C. trachomatis EBs
C. trachomatis EBs at a MOI of 0.05 were pre-incubated in DMEM supplemented with 10% FBS, in the absence or presence of OOP in NaDES at the highest effective concentration, for 1 h at 37 • C in a humidified atmosphere with 5% CO 2 . Subsequently, 100 µL aliquots of each solution containing C. trachomatis EBs were placed on a polylysine-covered glass coverslip (12 mm diameter) and put to dry at room temperature for 3 h. Samples were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 1.5 h, post-fixed with 1% OsO 4 in the same buffer for 2 h, dehydrated through a graded ethanol series, critical point dried with CO 2 (CPD 030 Balzers device, Bal-Tec, Balzers, Pfäffikon, Switzerland), and gold coated by sputtering (SCD040 Balzers device, Bal-Tec). Samples were examined with a field emission gun scanning electron microscope (SEM-FEG, Quanta 200 Inspect, FEI Company, Eindhoven, The Netherlands).

Transmission Electron Microscopy of Pre-Treated C. trachomatis EBs
C. trachomatis EBs at an MOI of 0.05 were pre-incubated in DMEM supplemented with 10% FBS, in the absence or presence of OOP in NaDES at the highest effective concentration, for 1 h at 37 • C in humidified atmosphere with 5% CO 2 . Subsequently, C. trachomatis EB suspensions were used to infect McCoy cell monolayers, grown on glass coverslip in 24-well cell-culture trays, by centrifugation at 750× g for 30 min at room temperature. The cells were, then, washed with DPBS to remove non-internalized C. trachomatis EBs and newly incubated in DMEM supplemented with 10% FBS. After 2 h of incubation at 37 • C in humidified atmosphere with 5% CO 2 , cells were fixed in 2% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M sodium cacodylate buffer containing 3 mM CaCl 2 and 0.1 M sucrose (pH 7.4), at room temperature for 30 min, and stored at 4 • C. After waiting overnight, the fixed cells were scraped, washed in 0.15 M sodium cacodylate buffer containing 3 mM CaCl 2 (pH 7.4) and centrifuged. The pellets were resuspended and postfixed in 2% osmium tetroxide in 0.07 M sodium cacodylate buffer containing 1.5 mM CaCl 2 (pH 7.4) at 4 • C for 2 h, dehydrated through graded ethanol concentrations and embedded in Epon 812 resin (Electron Microscopy Science, Fort Washington, PA, USA). Ultrathin sections, obtained with a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany), were contrasted with UranyLess EM Stain (Electron Microscopy Sciences, Hatfield, PA, USA) and lead citrate, and examined with a Philips 208S transmission electron microscope (FEI Company, Eindhoven, The Netherlands).

Statistical Analysis
All values are expressed as means ± standard deviation (SD) of two to four replicates from at least two independent in vitro experiments. Comparisons of means were performed using a two-tailed Student t-test for independent samples. The single or multiple inference significance level was set to 5%. All statistical calculations and graphs were performed in the software Excel (Microsoft, Redmond, WA, USA, version 2302, build 16130.20332).