WO2025172967A1 - Surfactant-based hydrogel, methods and uses thereof - Google Patents

Abstract

The present disclosure relates to a topical pharmaceutical composition for use in the treatment of skin and/or mucosal infections or disorders, comprising a pharmaceutically effective amount of a dodecylpyridinium salt and a suitable hydrogel.

Description

SURFACTANT-BASED HYDROGEL, METHODS AND USES THEREOF

TECHNICAL FI ELD

[0001] The present disclosure relates to a novel antibiotic-free surfactant-based hydrogel, preferably a cationic surfactant-based hydrogel, for use in the prevention and treatment of skin and/or mucosal infections or disorders.

BACKG ROUN D

[0002] Healthy skin offers a natural barrier to invasion by pathogens, namely bacteria. Hence, impairment of this barrier, may make an individual susceptible to infection. Thus, physical traumas in the form of abrasions, penetrations, burns, pre-existing dermatoses with impaired barrier states, diabetes mellitus, and various congenital and acquired immunodeficiency syndromes, among others, all can potentially lead to cutaneous bacterial infections.

[0003] Staphylococcus aureus and group A Streptococci are the two most frequently encountered pathogens causing primary and secondary infections of the skin. Topical antibiotics are commonly used for the prevention and treatment of skin infections. Nonetheless, the rise of multi-resistant bacteria, as Methicillin-resistant Staphylococcus aureus (MRSA), has reduce the efficacy of a range of antibiotics of different classes and highlights the importance of the development of new strategies and products to fight topical bacterial infections.

[0004] Mucosal infections, namely sexually transmitted infections (STIs) and urogenital perinatal infections (UGls) are major public health issues, particularly in developing countries, where access to healthcare services is more limited.^1,2 As most of these infections are asymptomatic or have only mild symptoms, they often go undetected, contributing to their persistence and transmission. According to the World Health Organization (WHO), more than one million curable STIs are acquired every day among people aged 15-49 years, with more than half of the cases attributable to Chlamydia trachomatis and Neisseria gonorrhoeae. If left untreated, bacterial STIs can cause pelvic inflammatory disease, infertility, and ectopic pregnancy in women, as well as perinatal or congenital infections in infants born to infected mothers. Co-infection with other major STIs (like HIV, herpes, and HPV) is common and may promote synergistic effects on transmission and disease severity. Despite treatment being available for most bacterial STIs, antimicrobial resistance (AMR) has increased in the last years, reducing treatment options. This is particularly critical for N. gonorrhoeae, which is currently resistant to almost all classes of antimicrobials available for treatment, and has already been recognized by the WHO and the Center of Disease Control (CDC) as a "superbug" that may soon be untreatable. Perinatal vertically transmitted UGI caused by the Gram-positive Streptococcus agalactiae (also known as group B Streptococcus or GBS) is the main cause of early neonatal sepsis via maternal rectovaginal colonization. According to a recent WHO report, in 2020, S. agalactiae was responsible for 91,000 newborn deaths and left 40,000 infants living with neurological impairment. Recently, multidrug resistant strains of S. agalactiae have been detected and are increasing worldwide, which in part may be due to the frequent (and sometimes unnecessary) intrapartum antibiotic prophylaxis to which all carrier-mothers are subjected at delivery, mainly in rich/developed countries.

[0005] In the past, N. gonorrhoeae was readily treated with antibiotics. However, over time, this bacterium has developed resistance to almost all commonly used antibiotics. Consequently, there is now only one remaining effective class of antibiotics, cephalosporins, for treating this infection. Unfortunately, it has been witnessed a global emergence of cephalosporin-resistant gonorrhoea. Furthermore, resistance is also spreading due to the dual treatment that combines cephalosporins and azithromycin, affecting regions like the United States and the United Kingdom. In consequence, N. gonorrhoeae is becoming increasingly difficult to treat. According to the CDC 2021 guidelines, for cephalosporin-resistant N. gonorrhoeae strains, the infectious disease should be tackled with a dual treatment with single doses of IM gentamicin plus oral azithromycin.

[0006] The continuous rise in drug resistance, together with the lack of effective vaccines , makes the development of novel preventive and therapeutic measures urgently needed.² In the last decades, topical microbicides have been appearing as attractive options against STIs as they can be applied intravaginally or rectally under diverse formulation types (gels, creams, films, rings, sponges or suppositories) with or without spermicidal activity, greatly empowering women and men who have sex with men (MSM) to protect themselves. However, so far, current topical microbicides have shown only modest to no evident effect on STI acquisition. In contrast to the latest developed microbicidal agents that only have a narrow spectrum against viruses (mostly HIV), the first-generation microbicides were surfactant-based due to their well-known broad-spectrum antimicrobial activity, low price, chemical stability, and non-demanding storage requirements. Despite being extensively used as disinfectants and preservatives, they failed as prophylactic antiseptics against STIs of viral origin in Phase 3 clinical trials on human subjects due to the unnecessarily high concentrations (at or above the surfactant critical micelle concentration - CMC) used in those trials,^{3 5} which compromised their further testing.

[0007] These facts are disclosed to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0008] The present disclosure relates to a novel antibiotic-free cationic surfactant-based hydrogel for use in the prevention and treatment of skin and/or mucosal infections or disorders, for instance, in the prevention and treatment of sexually transmitted infections (STIs) and perinatally transmitted infections (PTIs).

[0009] The present disclosure relates to the microbicide activity of a topical composition comprising a hydrogel and a surfactant - dodecylpyridinium salt - for use as an agent to prevent and treat skin and/or mucosal infections or disorders. Surprisingly, the topical composition of the present disclosure showed to be safe, not affecting other bacteria of the microbiome (e.g., vaginal microbiome) and do not cause inflammation. Moreover, the composition is stable, allows a sustained release, it is not cytotoxic and do not potentiate bacterial resistance. The composition of the present disclosure rises as a novel antibiotic- free treatment against strains involved in skin and/or mucosal infections, namely Neisseria gonorrohoeae, Staphylococcus aureus including AMR strains, Pseudomonas aeruginosa, Streptococcus agalactiae among others Streptococcus.

[0010] The composition of the present disclosure may be applied as a topical composition externally onto the skin or in mucous membranes. Examples of routes of delivery to mucosal surfaces are respiratory, oral, vaginal, and rectal.

[0011] The present disclosure relates to a safe and efficacious surfactant-based hydrogel that provide treatment and protection against infections of bacterial origin. The composition water-based scaffold of the present application comprises N-dodecylpyridinium salt, preferably N-dodecylpyridinium bromide (C₁₂PB). It was surprisingly found that the hydrogel developed, showed to be effective in fighting bacterial infections, namely N. gonorrhoeae, and at the same time demonstrated a significantly lower potential for inducing antimicrobial resistance compared to traditional antibiotics and that prolonged exposure at sub-minimum inhibitory concentrations (sub-MIC) did not adversely impact bacterial susceptibility to antibiotics. Notably, this occurred without triggering any toxic or inflammatory responses in polarized epithelial cells.

[0012] The N-dodecylpyridinium salt was incorporated into a cellulose-based hydrogel formulation. It was found that the hydrogel developed offer a sustained release of the surfactant, thereby enhancing its biocompatibility while simultaneously maintaining its antibacterial activity. Rigorous pre-clinical safety assessments were carried out and evaluated the anti-bacterial efficacy of the C₁₂PB-hydrogel formulation in vitro and in vivo. The positive outcomes of these tests and the absence of toxicity and inflammation affirmed the efficacy and safety of the microbicidal hydrogel/topical formulation of the present disclosure. [0013] Regarding its use in vaginal infections, the topical composition of the present disclosure assures a steady concentration of the drug in the vaginal cavity, which minimizes the need for multiple doses. This innovative strategy presents a significant advancement in the field, providing a promising tool in the fight against direct-contact transmitted infections.

[0014] The pharmaceutical composition of the present disclosure proved to be safe in vivo in a wildtype mouse model of mice infected with Neisseria gonorrhoeae.

[0015] Importantly, the active molecule used in this formulation, N-dodecylpyridinium salt , preferablyC₁₂PB (N-dodecylpyridinium bromide, CAS No. 104-73-4), is endowed with a dual attractive characteristic: I) a very low or slow potential of inducing antimicrobial resistance, while it lacks any cross-resistance effect that may adversely affect bacterial susceptibility to antibiotics and ii) a noninflammatory profile, at subtoxic concentrations towards host cells, preventing further infectious diseases.

[0016] The N-dodecylpyridinium salt hydrogel of the present disclosure, preferably C12PB hydrogel, is characterized by enhanced properties, facilitating a synergistic interaction between the hydrogel matrix and the N-dodecylpyridinium compound. This interaction enables a controlled release and localized distribution of the active compound, ensuring a sustained, effective, and safe concentration for the treatment and prevention of bacterial infections over time. In a preferred embodiment, said infections are N. gonorrhoeae infections. The N-dodecylpyridinium hydrogel of the present disclosure exhibit enhanced biocompatibility, while maintaining antibacterial activity and a reduced potential to trigger inflammatory responses in vitro and in vivo.

[0017] A critical aspect of the hydrogel of the present disclosure is its viscosity. The gel-like consistency of the developed hydrogel is crucial for the controlled release and localized distribution of the active compound, as well as for maintaining vaginal epithelial barrier integrity, in case of vaginal application.

[0018] The composition of the present disclosure can be applied locally to the vaginal mucosa. Notably, the intravaginal application of C₁₂PB-loaded hydrogel did not cause changes in the vaginal diversity microbiome in mice, suggesting that vaginal health is preserved. This result is of utmost importance since an imbalance of vaginal flora and subsequent complications can have major drawbacks for tackling infectious diseases and also for the compliance with the formulation of the present disclosure. Importantly, a single application of C₁₂PB-loaded hydrogel/topical composition was able to reduce gonorrhoea in a mouse model of N. gonorrhoeae infection. All this, allied with the fact that C₁₂PB possesses a very low/slow potential for inducing AMR compared with traditional antibiotics and does not adversely affect bacterial susceptibility to antibiotics, assures the applicability in the long run of this microbicide formulation. [0019] In an embodiment, the topical composition is for vaginal use, wherein it showed to be effective against Sreptococcus agalactiae and Neisseria gonorrohoeae (N.gonorrohoeae), including AMR strains. The composition can also be used for rectal administration.

[0020] In a preferred embodiment, the hydrogel of the present disclosure is colourless and odourless.

[0021] The intravaginal administration of the surfactant-hydrogel of the present disclosure did not trigger an inflammatory response neither epithelial disruption. The hydrogel of the present disclosure is a microbiome-friendly vaginal microbicide, as the diversity of vaginal microflora seems not to be affected in vivo.

[0022] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. More specifically, pharmaceutically acceptable refers to a material, compound, or composition which is suitable for use in contact with the skin or mucosa. Pharmaceutically acceptable materials are known to those of ordinary skill in the art.

[0023] As used herein, the terms "treatment" or "treating" are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0024] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. [0025] An aspect of the present disclosure relates to a topical pharmaceutical composition for use in the treatment of skin and/or mucosal infections or disorders wherein said topical composition comprises a pharmaceutically effective amount of a dodecylpyridinium salt and a suitable hydrogel; wherein the topical pharmaceutical composition comprises a viscosity ranging from 1 to 20 Pascal seconds (Pa.s) when measured at a shear rate of 25s ¹ and 37 °C.

[0026] In a preferred embodiment, the viscosity of the composition ranges from 1 to 15 Pascal seconds (Pa.s); preferably ranges from 1 to 10 Pascal seconds (Pa.s); when measured at a shear rate of 25s ¹ and 37 °C.

[0027] In the state of the art, the viscosity may be measured by many methods. In the present disclosure the viscosity measurement was carried out as measured on Physica MCR-501 rheometer from Anton Paar running the RheoPlus Software. Samples were tested using 25 mm parallel plates with a 1 mm gap, set to a temperature of 37 °C. The viscosity of the solution was measured in response to increasing shear rate from 0.01 to 1000 s'¹.

[0028] The suitable hydrogel of the pharmaceutical composition described in the present disclosure is defined as a biocompatible polymer capable of absorbing and retaining water, wherein the hydrogel is a biocompatible hydrogel, preferably the hydrogel is selected from the group consisting of: collagen, polyvinyl alcohol, polysaccharides, cellulose derivatives and biocompatible organic polymers and copolymers, preferably wherein the biocompatible hydrogel is selected from the group consisting of alginate, RGD-modified alginate, amylase, amylpectin, cellularose, chitosan, collagen, dextran, fibrin, gelatin, glycogen, heparin, hyaluronic acid, poly(acrylamide), poly(P-aminoester), poly(caprolactone), matrigel, multi-arm polyethylene glycol, poly-hydroxyethyl acrylate, poly(hydroxyethyl methacrylate), poly(N-isopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolic acid), oligo(poly(ethylene glycol)fumarate), poly(vinyl alcohol), poly(vinyl acid), and combinations thereof; and/or comprising one or more pharmaceutically acceptable excipients selected from the group consisting of: solvents, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilisers, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, anti-foaming agent, viscosity modifier, sweeteners and combinations thereof; and/or

[0029] The hydrogel is designed to exhibit biocompatibility, stability, and controlled release properties. The hydrogel may be composed of natural or synthetic polymers, or a combination thereof, and is tailored to accommodate the therapeutic agent dodecylpyridinium bromide. The formulation provides a sustained and controlled release of the pharmaceutical compound, enhancing its bioavailability and therapeutic efficacy. [0030] In a preferred embodiment, the dodecylpyridinium salt is selected from the list consisting of: dodecylpyridinium chloride (Cas No. 104-74-5); dodecylpyridinium iodide (Cas No. 3026-66-2); dodecylpyridinium bromide (Cas No. 104-73-4). More preferably, the dodecylpyridinium salt is dodecylpyridinium bromide.

[0031] In a preferred embodiment, the amount of dodecylpyridinium salt is at least 375 μM and below the dodecylpyridinium salt critical micelle concentration (CMC). More preferably, the amount of dodecylpyridinium salt is less than 1000 μM. Even more preferably, the amount of dodecylpyridinium salt ranges from 375-1000 μM; preferably 400-900 μM; more preferably 600-800 μM.

[0032] In a preferred embodiment, the suitable hydrogel comprises a biocompatible polymer selected from a list consisting of: cellulose derivatives; polysaccharides; alginates; chitosans; polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid; gellan gum; carbopol; polyethylene glycol (PEG); acrylamide; hyaluronic acid; collagen, and biocompatible organic polymers and copolymers, dextran, fibrin, gelatin, glycogen, heparin, poly(P-aminoester), poly(caprolactone), matrigel, polyhydroxyethylacrylate, poly(hydroxyethyl methacrylate), poly(Nisopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolicacid), oligo(poly(ethylene glycol)fumarate), poly(vinyl acid), and/or biocompatible organic polymers andcopolymers, or mixtures thereof. More preferably, the the polymer is a cellulose-derivative polymer. Even more preferably, the cellulose derivative is selected from hydroxyethylcellulose, hydroxypropylmethylcellulose, or copolymers or mixtures thereof; preferably hydroxypropylmethylcellulose.

[0033] In a preferred embodiment, the hydrogel comprises 1-10 % (w/w) of polymer; preferably 1-5 % (w/w); more preferably 1-3 % (w/w).

[0034] In a preferred embodiment, the composition of the present disclosure further comprises an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a cell, a buffer, a growth factor, a second suitable hydrogel or combinations thereof.

[0035] Another aspect of the present disclosure relates to the composition herein described for use in the prevention or treatment of skin and/or mucosal infections or disorders caused by a bacterium.

[0036] Another aspect of the present disclosure relates to the composition herein described for use in the prevention or treatment of sexually transmitted infections and/or perinatal infections caused by a bacterium.

[0037] In a preferred embodiment, the bacterium is selected from the group consisting of: Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycoplasma genitalium, Gardnerella vaginalis, Treponema pallidum, Haemophilus ducreyi, Klebsiella granulomatis (or Calymmatobacterium granulomatis) or mixtures thereof. In a more preferred embodiment, the bacterium is Neisseria gonorrhoeae. [0038] In an embodiment, the composition is administered topically to a skin area or to a mucosal area. In a preferred embodiment, the mucosal area is selected from the group consisting of vaginal, rectal and buccal cavities.

[0039] In an embodiment, the said mucosal area is on the vulva, perianal, the lining of the anus and on the penis.

[0040] In a preferred embodiment, the composition is an intravaginal composition.

[0041] In a preferred embodiment, the composition is a sustained-release topical pharmaceutical composition.

[0042] In an embodiment, the composition is a single dose composition or a multiple dose composition.

[0043] Another aspect of the present disclosure relates to the use of the topical composition herein described for the manufacture of a medicament for use in the treatment of skin and/or mucosal infections or disorders.

[0044] Another aspect of the present disclosure relates to a method for treating or preventing skin and/or mucosal infections or disorders, the method comprising administering the topical pharmaceutical composition herein described to the subject.

[0045] Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

BRI EF DESCRI PTION OF THE DRAWI NGS

[0046] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0047] Figure 1 a) Genetic diversity (i.e., the emergence of SNPs or indels) observed among the fully sequenced gonococcal populations evolved under C₁₂PB pressure (detailed in Table 8), with unfilled dashed circles representing minor variants (frequencies >20%-<50%), filled dashed circles representing major variants (frequencies >50%-<90%) and filled solid circles representing fixed mutations (frequencies >90%). Type of mutation is identified as follows: synonymous (square), missense (triangle) and frameshift (circle).

[0048] Figure 1 b) N. gonorrhoeae susceptibility to multiple antibiotics after prolonged exposure to C₁₂PB (detailed in Table 6). Graphs show, for each antibiotic, the MIC values determined for the original isogenic clone (triangle) and respective gonococcal populations (circles) from the latest continuous passage of each selective pressure assay (i.e., at P126 for N. gonorrhoeae). Dotted lines represent the current EUCAST breakpoints for each antibiotic for each microorganism. Shadowed regions represent MIC values that fall within a range rather than being exactly determined. For simplification purposes, bacterial populations were designated according to the 24-well plate, with Al-Dl representing positive controls (without compound) and A5-D5/A6-D6 referring to bacterial populations under compound selective pressure.

[0049] Figure 1 c) S. agalactiae susceptibility to multiple antibiotics after prolonged exposure to C₁₂PB (detailed in Table 7). Graphs show, for each antibiotic, the MIC values determined for the original isogenic clone (triangle) and respective streptococcal populations (circles) from the latest continuous passage of each selective pressure assay (i.e., at P126 for N. gonorrhoeae and P56 for S. agalactiae). Dotted lines represent the current EUCAST breakpoints for each antibiotic for each microorganism. Shadowed regions represent MIC values that fall within a range rather than being exactly determined. For simplification purposes, bacterial populations were designated according to the 24-well plate, with Al-Dl representing positive controls (without compound) and A5-D5/A6-D6 referring to bacterial populations under compound selective pressure.

[0050] Figure 2 a)- C₁₂PB in vitro toxicity and inflammation towards polarized epithelial Caco-2 cells. Minimum inhibitory concentration (MIC) of C₁₂PB against different strains of N. gonorrhoeae and S. agalactiae.

[0051] Figure 2 b) C₁₂PB surfactant toxicity profile towards polarized Caco-2 cells. The grey area represents the range of MICs for several strains of N. gonorrhoeae and S. agalactiae. The CMC of C₁₂PB (3900 μM) is represented by the dashed line. Cell viability was assessed by the MTT assay 24 h after the cells had been exposed to different concentrations of surfactants for 20, 60, 180 and 360 minutes. Cell viability is expressed as a percentage of the viability of control cells.

[0052] Figure 2 c) Inflammatory response profiles in Caco-2 polarized cells after exposure to the C12PB surfactant (3.9 μM, 25, and 39 μM). Early/acute (immediately after incubation with surfactant for 180 min) (i) and late (24 h post-surfactant removal) (ii). Data are presented as the mean ± SD of at least 3 independent experiments, each done in triplicate. Abbreviations: Below Detection Limit (BDL).

[0053] Figure 3 a) Hydrogel physicochemical properties characterization. Rheological properties of gel formulations, including i) viscosity as a function of shearing rates and ii) viscoelastic properties at 1 Hz.

[0054] Figure 3 b) SEM imaging of i) F4M, ii) F4M C₁₂PB (25 μM), iii) HEC and iv) HEC C₁₂PB (25 μM) hydrogel formulations.

[0055] Figure 3 c) Release profile of surfactant from the hydrogel network (50 μM), including HEC and F4M hydrogels, for 1440 min (1 day). Scale bar: 40 pm (b). [0056] Figure 4 a) In vitro evaluation of antibacterial activity, toxicity, and inflammation of C₁₂PB- containing hydrogels, i) Microbicidal activity of F4MC₁₂PB, HEC C₁₂PB, and C₁₂PB (50 μM) towards N. gonorrhoeae and ii) S. agalactiae bacteria.

[0057] Figure 4 b) i) Schematic of the indirect assay of F4M gel and C₁₂PB (50 μM) supernatant collection for incubation with ii) N. gonorrhoeae and iii) S. agalactiae bacteria.

[0058] Figure 4 c) Viability assay of i) Caco-2. Cell viability is expressed as a percentage of the viability of cells in only gel conditions (F4M and HEC) and untreated cells (C₁₂PB). Expression of inflammatory cytokines, namely (ii and iv) IL-8 and (iii and v) IL-6 in Caco-2 cells cultured with different gel formulations (ii and iii) after 3 h and 6 h of incubation and (iv and v) and 24 h (3 + 24 h, and 6 + 24h) after removal of the C₁₂PB -containing hydrogel from cell culture. Surfactant alone and empty vaginal gels were used as controls. Data are shown as mean ± SD (n=4; **p<0.01, unpaired t test).

[0059] Figure 5 a) In vivo degradation and biodistribution of hydrogel. Whole body in vivo degradation and biodistribution.

[0060] Figure 5 b) Organ biodistribution at 48h post-F4M hydrogel vaginal application.

[0061] Figure 5 c) In vivo FITC signal quantification.

[0062] Figure 5 d) FITC signal quantification by organ at 48h post F4M hydrogel vaginal application. Data are presented as mean ± SD (n=4; **p<0.01, unpaired t test).

[0063] Figure 6- In vivo inflammatory and epithelial disruption of vaginal tract tissue, a) i) Histological analysis of vaginal total score, ii) edema fibrosis score and iii) inflammatory infiltrate score after intravaginal administration of F4M gel containing C₁₂PB at different CMC concentrations (390-39000 μM). The values (scores) assigned for each of these lesions were as follows: 0 (no change) when no injury or the observed changes were within the normal range; 1 (minimum) when changes were sparse but exceeded those considered normal; 2 (light) when injuries were identifiable but with no severity; (3) moderate for significant injury that could increase severity; 4 (very serious) for very serious injuries that occupied most of the exceeded tissue. The score was used to evaluate epithelial integrity, epithelial vascular congestion, inflammatory infiltrate, and edema fibrosis in each formulation. These values were summed to obtain a total score and determine the overall histological health of the vaginal tract tissues as minimum 1-4; average 5-8; moderate 9-11 and severe 12-16. Thus, the total score reflects the cumulative effect of the hydrogel on tissue structure and immune response, offering insights into its potential for clinical use and the need for optimization to minimize tissue disruption and inflammation, b) histological evaluation of the vaginal tissue of anti-CD45. Data are presented as mean ± SD (n=3; ***p<0.001, **p<0.01 and *p<0.05, one-way ANOVA followed by a Tukey's test for multiple comparisons). [0064] Figure 7a) Efficacy of C₁₂PB-hydrogel towards an in vivo murine model of N. gonorrhoeae infection. Schematic illustration of microbiome evaluation.

[0065] Figure 7b) Impact of C₁₂PB-hydrogel (CMC/10 - 390 μM and CMC/5 - 780 μM) on composition of mice vaginal microbiota. The top graph shows the Shannon entropy of the different sample groups, with each bar representing one biological sample, while the bottom graph displays a principal component analysis.

[0066] Figure 7c) Schematics of the in vivo efficacy experiment.

[0067] Figure 7d) Effect of a single application of C₁₂PB-hydrogel (from 25 μM to 780 μM) on N. gonorrhoeae infected mice. Data are presented as mean ±SEM (n=5; **p<0.01 and *p<0.05, one-way ANOVA followed by a Tukey's test for multiple comparisons).

[0068] Figure 8 (a-b)- Schematic illustration of bacterial populations selected for whole-genome sequencing throughout each in vitro selective pressure assay. For both bacterial isolates, the conducted assays (see methods for details) are represented by different timelines gray colored per compound. Besides the N. gonorrhoeae (PT NG) and S. agalactiae (PT GBS) initial clones, all bacterial populations subjected to WGS (selected according to phenotypic data - see results section) are shown above each vertical line, corresponding to the respective continuous passage. For simplification purposes, bacterial populations were designated according to a 24-well plate, with Al-Dl representing positive controls (without compound) and A5-D5/A6-D6 referring to bacterial populations under compound selective pressure. Created with BioRender.com.

[0069] Figure 9 - Streptococcal growth curves in the presence of C₁₂PB (A) and erythromycin (B). Each graph represents the growth of PT GBS initial clone without (positive control) and under different concentrations (1/8 MIC, 1/4 MIC, 1/2 MIC, and MIC) of each compound. Negative controls (broth solely) are also shown. Values plotted are expressed as mean ± SD.

[0070] Figure 10 -LC-HRMS chromatogram of C₁₂PB at 50 μM and corresponding HRMS spectrum.

[0071] Figure 11 - In vivo inflammatory and epithelial disruption of vaginal tract tissue histological evaluation, a) Histopathology of vagina, cervix, uterus, and ovary tissue in Balb/c mice upon intravaginal administration of F4M gel and F4M gel containing C12PB at different CMC. b) Immunohistochemical images staining of CD45 of vaginal tissue, c) Immunohistochemical images staining of CD45 of cervix tissue. Scale bar: (a-vagina, cervix) 100 pm, (a-uterus, ovary) 500 pm, and (b-c) 50 pm. DETAI LED DESCRI PTION

Profiling surfactants towards bacteria response and cell survival

Tables 1-3 resumes the study of the analysis of the responses of pathogens and cell lines to surfactants. Abbreviations: Nonionic: Triton X-100 (TX-100), zwitterionic-N-dodecyl-N,N-dimethylammonium- propanesulfonate (DDPS). Anionic: sodium dodecyl sulfate (SDS). Cationic: n-Alkyl-N,N,N- trimethylammonium bromides (C_nTAB with n from 10 through 14), N-dodecylpyridinium bromide (C₁₂PB), and Dodecyl-N-benzyl-N,N-dimethylammonium (better known as Benzalkonium) bromide (C₁₂BZK) surfactants.

Table 1. Effect of the surfactant type and concentration on different pathogens. Concentrations are normalized with respect to the CMC of each surfactant at four hours after incubation.

Table 2. Lethal dose (LD) of surfactants at 9 h after incubation, followed by a 24-hour chase normalized by the CMC of each surfactant. Low LD/CMC values mean that the concentrations of a given surfactant that cause toxicity towards host cells occur at concentrations much below the CMC. MDCK and Caco-2 cells are polarized epithelial cell lines. HeLa is an epithelial-like cell line. FSDC is a dendritic cell line. Abbreviations: LD: lethal dose. CMC: critical micelle concentration. Nonionic: Triton X-100 (TX-100), zwitterionic-N-dodecyl-N,N-dimethylammonium-propanesulfonate (DDPS). Anionic: sodium dodecyl sulfate (SDS). Cationic: n-Alkyl-N,N,N-trimethylammonium bromides (CnTAB with n from 10 through 14), N-dodecylpyridinium bromide (C₁₂PB), and Dodecyl-N-benzyl-N,N-dimethylammonium (better known as Benzalkonium) bromide (C₁₂BZK) surfactants.

Table 3. LD50/minimum inhibitory concentration (MIC50) for the three (C12) n-alkyl-QAS analogues after incubation with mammalian cell lines and different pathogens. LD50 was obtained after 60 min of exposure to QAS

Table 4. LD50/MIC50 of surfactants for different clinical isolate samples. High LD50/MIC50 values mean that for a given surfactant, the concentrations necessary to kill host cells were much higher than those necessary to kill bacteria. As above, LD50/MIC50 were determined using fully polarized Caco-2 cells after 60 min of exposure to QAS.

[0072] Surfactants are known to indiscriminately impact cell membrane integrity at concentrations near the CMC.⁶ However, from the representatives of the different families of commercially available surfactants - nonionic (Triton X-100), zwitterionic [N-dodecyl-N,N-dimethylammonium- propanesulfonate, (DDPS)], anionic [sodium dodecyl sulfate (SDS)], and cationic quaternary ammonium surfactants [QAS: alkyl-N,N,N-trimethylammonium bromides (CnTAB, n = 10 to 14), C₁₂PB, and dodecyl- N-benzyl-N,N-dimethylammonium bromide (C₁₂BZ)] tested, QAS may work as selective bactericides against several direct transmitted pathogens when used at concentrations lower than the CMC (Table 1). QAS surfactants were also toxic towards polarized epithelial cells (MDCK and Caco-2), epithelial-like (HeLa) and dendritic (FSDC) cells at concentrations far below their CMC (Low LD/CMC) (Table 2). On the contrary, Triton X-100, DDPS and SDS were toxic to all cell types tested at concentrations around their CMC (High LD/CMC), suggesting a non-selective mode of action involving cell membrane destabilization and/or destruction. Furthermore, QAS with short n-alkyl groups (C10-C12) were found to be more discriminatory bactericides when compared to its analogue with longer n-alkyl groups (C14) at sub-toxic concentrations for host cells (Table 2 and Table 3). QAS selectivity was also dependent on the chemical nature of the polar head group.

[0073] From a set of three (C12) n-alkyl-QAS analogues tested [TAB, benzyl-dimethylammonium bromide (BZK), and pyridinium bromide (PB)], surprisingly C₁₂PB exhibited the highest LD50/MIC50 value, meaning that the concentrations necessary to kill host cells were much higher than those necessary to kill bacteria (Table 3). The same tendency was observed for different isolates of l\l. gonorrhoeae and S. agalactiae (Table 4). Altogether, these results indicate that C₁₂PB may work as bactericides at concentrations that are not harmful to host cells.

Evaluation of antimicrobial resistance acquisition by N. gonorrhoeae and S. agalactiae under selective pressure

[0074] It was evaluated the capacity of C₁₂PB to induce AMR against N. gonorrhoeae and S. agalactiae, which are representative examples of a sexually transmitted and urogenital-perinatal bacterial infection, respectively. For this evaluation, two clinical isolates from the stock culture collection of the STI Reference Laboratory at the Portuguese National Institute of Health were selected. While the N. gonorrhoeae isolate (PT NG), belonging to MLST ST-9363 and NG-MAST ST2992, was selected from a panel of gonococcal isolates previously tested against C₁₂PB,⁸ the chosen S. agalactiae isolate (PT GBS) is a CC19 (ST-19) / capsular-type V human clinical strain that belongs to one of the most predominant lineages in colonized pregnant women (Table 5).⁹

[0075] For both bacteria, the AMR acquisition was evaluated by independent in vitro selective pressure assays, where isogenic clones of each selected isolate were continuously propagated at the exponential growth phase, without and under sub-MIC conditions. As a proof-of-concept, the same evaluation was done using antibiotics previously recommended in the treatment of N. gonorrhoeae (azithromycin, AZT) and S. agalactiae (erythromycin, ERY), for which both PT NG and PT GBS are susceptible (Table 6 and Table 7), and that are known to induce antimicrobial resistance in these microorganisms.

[0076] For N. gonorrhoeae, isogenic clones were continuously propagated every 24 h without and under 1/8 MIC of C₁₂PB or AZT. Overall, the in vitro propagation assay under surfactant sub-MIC pressure encompassed 126 passages, while the assay under AZT exposure comprised a total of 74 passages. For C₁₂PB, from the initial eight replicates of the original clone subjected to surfactant's pressure, it were follow four bacterial populations (A5, A6, B6 and D6) until the end of the assay (Fig. 8a). In general, a relatively stable C₁₂PB MIC (25 μM) was observed throughout the assay, with all populations developing only a one-fold MIC increase after 41 continuous passages, which remained unchanged until the end (Table 13). Comparative genome analyses between 39 gonococcal populations that evolved without and under C₁₂PB pressure versus the original isogenic clone ("ancestral") allowed the identification of few fixed mutations (4-5 per bacterial population) falling on genes (or promoter regions) from diverse functional categories, like bacterial cell envelope, motility, transport and metabolism, or signal transduction (Fig. la and Table 8).

[0077] Nevertheless, none of these mutations were simultaneously shared by the four bacterial populations at the same passage, and heterogeneity was also observed among passages. To further assess the assay reproducibility showing the 1-fold MIC increase, which falls within the acceptable variation MIC range, it was performed a parallel experiment by restarting the whole selective pressure assay from passage 25 (the latest without phenotypic changes) (Fig. 8). The obtained results mirrored the ones described above. Indeed, not only duplication in C₁₂PB baseline MIC was seen for the following eight gonococcal replicates after a total of 52 passages (Table 14), but also the analysis of 24 genomes from two dispersed representative passages (P60 and P88) (Fig. 8) revealed, once again, the fixation of a few mutations heterogeneously per bacterial population and passage (Table 9).

[0078] Interestingly, most of the detected mutations were not coincident among the two assays, which likely points to a laboratory pressure association. C₁₂PB results contrast sharply with those obtained for gonococcal isogenic clones under AZT sub-MIC pressure, where the baseline MIC (0.38 pg/ml) doubled just after 14 passages, with bacterial populations with different levels of susceptibility to AZT emerging after that (Table 15). Such diversity was clearly visible, for instance, among the seven bacterial populations recovered at the end of the assay, for which MIC increases of 3.9-fold (1.5 pg/mL) (n=l), of 21.1-fold (8 pg/mL) (n=5), and of 31.6-fold (12 pg/mL) (n=l) were seen. While the low-level of resistance exhibited by the D6 gonococcal population was found to coincide with the appearance of G70D and A211V substitutions in RpID 50S ribosomal protein L4 and efflux pump component MtrE, respectively, from passage 36 onwards, the abrupt escalation in MIC seen in the remaining populations was correlated with the 23SrRNA C2597T mutation, known to confer moderate-level resistance to AZT in i\i. gonorrhoeae. One population (C5) also exhibited an extra G297D mutation on the efflux pump component MtrC, but apparently without impacting the phenotypic MIC when compared with other gonococcal populations. Similar to C₁₂PB, several mutations associated with non-resistance settings (i.e., cell metabolism, sugar transport) (Table 10) and/or that emerged from continuous laboratory passaging were further found among the gonococcal populations evolved under AZT.

[0079] For the selected streptococcal isolate, in vitro selective pressure assays comprised a total of 56 passages, with isogenic clones continuously propagated every 12 h without and under 1/4 MIC of C₁₂PB or ERY, based on the respective bacterial growth curves (Fig. 11). Overall, with the exception of one replicate (B6) exposed to ERY, all replicates were completely followed until the end of the assay. No change in C₁₂PB MIC (12.5 μM) was observed during the whole assay (Table 16), which was also confirmed at the genome level. Indeed, when compared to the initial isogenic clone, only two nonsynonymous mutations on the pulA (SAG0852) and relA (SAG1940) genes were differentially found in four out of the 28 fully sequenced streptococcal populations from distinct continuous passages (Table 11).

Even though the impact of these mutations is unknown, these genes code for type I pullulanase and (p)ppGpp synthetase II, which are known to be involved in the bacterial stringent response. In contrast, the ERY baseline MIC (0.125 pg/mL) doubled only after 7 passages and increased up to 16 times after 26 passages, reaching an incredibly high value (>256 pg/mL) from passage 44 onwards (Table 17). Likewise, for N. gonorrhoeae with AZT, streptococcal populations with different levels of susceptibility to ERY were detected throughout the assay. Comparative genome analyses revealed that streptococcal populations evolved under ERY pressure harboring, among others, mutations in the 50S ribosomal proteins L4 and L22, in the 23SrRNA (uracil(1939)-C(5))-methyltransferase and in the 23S rRNA, targets that were already implicated in resistance to this 14-member-ring macrolide (Table 17 and Table 12). However, the high-level of ERY resistance developed by some bacterial populations appears to be conferred by the presence of the A2063G mutation in at least four of the seven copies of the large subunit rRNA. The only exception occurred for the D5 population from P56 that, besides a R92H change in protein L22 also present in several populations from diverse passages, contains a unique Q75R mutation immediately adjacent to a highly conserved stretch of the protein L4, in which variability was already demonstrated to increase >4-fold MICs of 14-, 15- and 16-membered macrolides and streptogramin B.

NA -not applicable.

Table 6. Antimicrobial susceptibility testing of N. gonorrhoeae original clone and final populations. PenG - Penicillin; P-LACT - p-lactamase; CRO - Ceftriaxone; CIP - Ciprofloxacin; SPT - Spectinomycin; TET - Tetracycline; CFM - Cefixime; GEN - Gentamicin; AZM - Azithromycin. Antibiotic susceptibility was tested by the Breakpoint Method. Minimum inhibitory concentration (MIC) values are expressed at mg/L. a Due to the lack of an established breakpoint, gentamicin susceptibility was classified for analysis purposes based on interpretive results from previous studies: as susceptible when MIC < 4 mg/L, and resistant when MIC > 16 mg/L, according to Boiko et al., 2019; APMIS, 127(7):503-509 (doi:10.1111/apm.12948) and Brown et al., 2010; Sex Transm Dis., 37(3) :169-172 (doi:10.1097/OLQ.0b013e3181bf575c). b Antibiotic phenotype was interpreted according to the EUCAST Clinical Breakpoint Tables v. 13.0 (valid from 2023-01-01). S-susceptible; R-resistant, and l-Susceptible, increased exposure. * Positive control (bacterial populations without C₁₂ PB subletal exposure).

Table 7. Antimicrobial susceptibility testing of S. agalactiae original clone and final populations. AMX - Amoxicillin, PenG - Benzylpenicillin, CLI - Clindamycin, ERY -

Erythromycin, MXF - Moxifloxacin, LVX - Levofloxacin, LZD - Linezolid, NIT - Nitrofurantoin, TEC - Teicoplanin, TET - Tetracycline, TGC - Tigecycline, SXT - Trimethoprimsulfamethoxazole, VAN - Vancomycin. AST was based on the AST-P586 card of VITEC2. Minimum inhibitory concentration (MIC) values are expressed at mg/L. The susceptibility to AMX is inferred from the benzylpenicillin susceptibility, b For SXT, MIC is expressed according to the trimethoprim:sulfamethoxazole ratio of 1:19. c Antibiotic phenotype was interpreted according to the EUCAST Clinical Breakpoint Tables v. 13.0 (valid from 2023-01-01). S-susceptible; R-resistant, and l-Susceptible, increased exposure. * Positive control (bacterial populations without C12PB sublethal exposure).

Table 8. Mutations found in Neisseria gonorrhoeae populations under C₁₂PB selective pressure, fs - frameshift variant; * - premature stop gain; O - present mutation; § - ll li i t t 10 d k / t d

Fs: frameshift variant; * premature stop gain; (n) - n^° of copies; O: present mutation; §: allelic mixture; +<10 reads

Table 10. Mutations found in Neisseria gonorrhoeae populations under AZT selective pressure, (n) - n.⁵ of copies; O - present mutation; § - allelic mixture; + - <10 reads

Table 11. Mutations found in Streptococcus agalactiae populations under C₁₂PB selective pressure. O - present mutation; § - allelic mixture; t - <10 reads

Table 12. Mutations found in Streptococcus agalactiae populations under erythromycin selective pressure, (n) - n.⁵ of copies; O - present mutation; § - allelic mixture; - not sequenced

Table 13. Antimicrobial resistance (AMR) characterization of N. gonorrhoeae and S. agalactiae isogenic clones over time. MIC evolution of gonococcal populations continuously propagated without and under exposure to 1/8 MIC of C12PB (25 μM).

[0080] Table 14. MIC evolution of gonococcal populations continuously propagated without and under exposure to 1/8 MIC of C₁₂PB. This constitutes a parallel assay (from P25 to P105) of the one described in Table 13 and aimed at assessing the reproducibility of the obtained data, which showed a one-fold MIC increase (see methods for details). For simplification purposes, gonococcal populations were designated according to the 24-well plate with Al-Dl representing positive controls (without C₁₂PB) and A5-D5/A6-D6 referring to bacterial populations under C₁₂PB selective pressure. Passages marked with an asterisk (*) enroll bacterial populations selected for whole-genome sequencing. The black dashed line represents the C₁₂PB baseline MIC.

Table 15. Phenotypic and genotypic characterization of AMR acquisition by /V. gonorrhoeae isogenic clones during antibiotic selective pressure. MIC evolution of bacterial populations continuously propagated without and under AZT 1/8 MIC (for N. gonorrhoeae). For simplification purposes, bacterial populations were designated according to the 24-well plate, with Al-Dl representing positive controls (without compound) and A5-D5/A6-D6 referring to bacterial populations under compound selective pressure. Passages marked with an asterisk (*) enroll bacterial populations selected for whole-genome sequencing.

Table 16. MIC evolution of streptococcal populations continuously propagated without and under exposure to 1/4 MIC of C₁₂ PB (12.5 μM ).

Passages marked with an asterisk (*) enroll bacterial populations selected for whole-genome sequencing.

Table 17. Phenotypic and genotypic characterization of AMR acquisition by 5. agalactiae isogenic clones during antibiotic selective pressure. MIC evolution of bacterial populations continuously propagated without and under ERY 1/4 MIC (for S. agalactiae). For simplification purposes, bacterial populations were designated according to the 24-well plate, with Al-Dl representing positive controls (without compound) and A5-D5/A6-D6 referring to bacterial populations under compound selective pressure. Passages marked with an asterisk (*) enroll bacterial populations selected for whole-genome sequencing.

[0081] Unlike antibiotics, little attention has been paid to antimicrobial resistance to disinfectants. Both the lack of epidemiological cut-off (ECOFF) values and the absence of standardized methods for susceptibility testing hamper the association between disinfectant exposure and the appearance of antimicrobial resistance. By using an in vitro selective pressure approach that accurately mimics natural evolution in several pathogens, the inventors of the present disclosure surprisingly found that C₁₂PB displays a very low, or at least slow, potential of inducing AMR or decreased susceptibility after prolonged usage when compared to traditional antibiotics (used as proof-of-concept) for N. gonorrhoeae and S. agalactiae. Indeed, although gene expression was not evaluated, the lack of common genetic mutations acquired by gonococcal and streptococcal populations exposed to C₁₂PB corroborates the observed relatively stable baseline MIC (maximum one-fold increase).

[0082] Overall, together with its strong bactericide activity, it is herein demonstrated the improved properties of C₁₂PB for use as an effective antimicrobial agent, in particular against sexually transmitted infections and urogenital perinatal infections.

Evaluation of antibiotic cross-resistance

[0083] Given the potential implications of the repeated exposure of sub-minimum inhibitory concentrations (sub-MIC) of QAS in inducing bacterial stress responses and subsequent emergence of bacteria with reduced susceptibility to antibiotics, a comprehensive investigation of these effects regarding C₁₂PB was performed. Therefore, in order to evaluate whether prolonged usage of C₁₂PB at sub-MIC conditions may negatively impact N. gonorrhoeae and S. agalactiae susceptibility to traditional antibiotics, gonococcal and streptococcal populations from the latest continuous passage (P126 and P56, respectively) without and under C₁₂PB sub-MIC, were subject to AST. This knowledge is essential for assessing the safety and efficacy of C₁₂PB as a potential antimicrobial option against sexually transmitted infections and urogenital infections.

[0084] For both bacteria (Table 5 and Table 6), it was found no significant differences in antimicrobial susceptibility among the bacterial populations exposed to C₁₂PB and those propagated without C₁₂PB pressure that served as positive controls. Also, no differences were found when compared to the respective original isogenic clones. This lack of substantial variation was evidenced by either a slight decrease, a stabilization (the vast majority), or a maximum of one-level increase in the MIC value for each antibiotic tested, yielding no alteration of the original AMR profile (see Fig. lb-c). Overall, all N. gonorrhoeae populations displayed reduced susceptibility to both benzylpenicillin and gentamicin as well as low-level resistance to tetracycline, while being susceptible to AZT, ciprofloxacin, spectinomycin, ceftriaxone and cefixime. 5. agalactiae populations were susceptible to amoxicillin, benzylpenicillin, clindamycin, erythromycin, moxifloxacin, levofloxacin, linezolid, nitrofurantoin, teicoplanin, tigecycline, trimethoprim-sulfamethoxazole and vancomycin, but phenotypically resistant to tetracycline. These results were further confirmed at the genomic level (Table 8 and Table 11), with all gonococcal and streptococcal populations (/.e., original clones and the ones evolved under and without C₁₂PB) exhibiting the same AMR genetic determinants (100% identical).

[0085] Altogether, these results showed that, for both microorganisms, prolonged exposure to C₁₂PB sub-MIC does not adversely affect bacterial susceptibility to antibiotics. This apparent lack of cross-resistance has significant implications as it may reduce the putative emergence of, or even worsening, bacterial resistance to antibiotics.

Inflammatory profile of polarized epithelial cells exposed to C₁₂PB

[0086] Besides a lack of antimicrobial resistance, a good microbicide must be safe for the host. Thus, the first criterion to be considered in the evaluation of the safety of topical microbicides, namely topical vaginal microbicides, is their toxic and inflammatory behaviour toward the cervicovaginal mucosa. On ethical grounds, any such evaluation should begin with an in vitro screening before using animal models and human studies. To this end, it was assessed in vitro the potential toxicity and inflammation induced by C₁₂PB within the range of the MICs determined for N. gonorrhoeae and S. agalactiae isolates (Fig. 2a). The present assessment involved performing toxicity curves in Caco-2 columnar epithelial cells, a cell line that, although not of vaginal origin, is derived from mammalian columnar epithelia and can be grown to a completely confluent and polarized state with relatively non-leaky tight junctions, closely resembling the characteristics of the vaginal columnar epithelium of the cervix, which is the primary site of damage in the use of surfactants. ¹⁰ The results showed that cell viability remained largely unchanged at C₁₂PB concentrations within the MIC range for several N. gonorrhoeae and S. agalactiae isolates, specifically between 6.25 μM (CMC/616) and 25 μM (CMC/154), as illustrated in Fig. 2b. However, concentrations above 25 μM and longer incubation periods (180 and 360 min) began to show a decline in cell viability compared to control cells. Notably, the 25 μM concentration corresponds to the highest MIC for both N. gonorrhoeae and S. agalactiae isolates (Fig. 2a).

[0087] Inflammation is typically characterized by elevated levels of cytokines and chemokines in the genital tract. Therefore, it was assessed a set of biomarkers commonly used in vaginal microbicide studies (IL-8, IL-6, TNF-α, IL-1β, MCP-1/CCL2, MIP-1/CCL3, and IL-10) to evaluate inflammation. Quantification was conducted on Caco-2 cells supernatants exposed to C₁₂PB concentrations both below and above the MICs of the N. gonorrhoeas and S. agalactiae isolates. C₁₂PB concentrations used were CMC/1000 (3.9 μM), CMC/154 (25 μM), and CMC/100 (39 μM) as displayed in Fig. 2c. After exposure to C12PB surfactant for 180 min, it was measured the inflammatory biomarkers immediately after incubation with the surfactant (early/acute response, Fig. 2c-i) and 24 hours post-surfactant removal (late response, Fig. 2c-ii). Interestingly, the levels of IL-6, TNF-a, IL-1β, MCP-1/CCL2, and IL-10 in Caco-2 cells were below the detection limit, both immediately after incubation and 24 hours post surfactant removal. Only IL-8 and MIP-1/CCL3 levels were detectable, but these levels were minimal and showed no variation between C₁₂PB- treated and untreated cells.

[0088] Thus, according with the results obtained in vitro, the utilization of this surfactant appears to be non-toxic and non-inflammatory within the investigated concentrations. Based on these results, it can conclude that C₁₂PB holds promise as a safe and effective topical microbicide for managing sexually transmitted infections and urogenital infections.

C₁₂PB-Hydrogel exhibits sustained release and antibacterial activity

[0089] The development of an improved hydrogel capable of releasing the therapeutic agent in a sustained, controlled manner, combining with adequate adherence to vaginal tissue and prolonged retention, would support an extended course of treatment. Moreover, if removal is required, it should be easily eliminated without causing harm to the vaginal tissue.

[0090] To design an optimized hydrogel, it was formulated two different C₁₂PB compositions utilizing cellulose-based polymers: Hydroxyethylcellulose at 1.25 weight percent (HEC, Natrosol, Ashland), and hydroxypropylmethylcellulose at 2 weight percent (Methocel F4M, Colorcon). These formulations were chosen due to their high solubility and adequate viscosity, making them a practical, comfortable, and acceptable prevention method, particularly against STIs in less- affluent regions (Fig. 3a). The gel-like consistency of the developed hydrogel is crucial for maintaining vaginal epithelial barrier integrity. The integration of C₁₂PB into the polymeric network did not impact the hydrogel's morphology and microstructure (Fig. 3b), ensuring the maintenance of the hydrogel's physical properties and performance.

[0091] Transwell cell culture inserts were used to evaluate surfactant release profiles. Briefly, the hydrogel containing the surfactant compound was placed in the upper part of the transwell membrane, and water was added to the well. At different time points, the supernatants (placed in the well) were collected and replaced with fresh solutions. In terms of drug release, while both cellulose-based gels provided sustained release of the C₁₂PB surfactant, the Methocel F4M formulation (F4MC₁₂PB) demonstrated superior release efficacy compared to the HEC formulation (HEC C₁₂PB) (Fig. 3c). Remarkably, the Methocel F4M hydrogel showed a complete release of C₁₂PB after only one day. Interestingly, the local delivery of the hydrogel into the vaginal tract allows a high concentration of surfactant at the target site while hindering any adverse effects on other regions (e.g., the gastrointestinal tract). These data reinforce the potential of Methocel F4M-based hydrogels as effective and practical delivery systems for C₁₂PB in the treatment and prevention of STIs and UGls.

[0092] To ensure that C₁₂PB microbicide efficacy was not impaired by the cellulose polymers, isogenic clones of i\i. gonorrhoeae and S. agalactiae were put in direct contact (direct assay) with the C₁₂PB-hydrogel formulations for several time points (Fig. 4a). In a general way, both formulations showed in vitro to be highly effective in decreasing bacterial growth. Both gel formulations containing 50 μM of C₁₂PB (final concentration of 25 μM) demonstrated an efficacy of around 100% for N. gonorrhoeae and S. agalactiae 2 h after incubation (Fig.4a). The bactericide effect was seen by the total absence of bacterial growth after 24 h of incubation. The results were similar to those obtained for C₁₂PB alone, which is aligned with the high release efficiency of C₁₂PB in the cellulose polymers (Fig. 2c), allowing us to conclude that the antimicrobial effectiveness of C₁₂PB is not affected when conjugated with the hydrogels. However, FM4-C₁₂PB was more efficient in killing the pathogens than HEC-C₁₂PB. This outcome may be related to the fact that C₁₂PB release was slower in HEC- than in FM4- formulations (Fig. 3c).

[0093] Additionally, for the F4M-containing surfactant (F4M-C₁₂PB), it was also assessed its in vitro microbicide efficacy by an indirect method (Fig. 4b). This method consists of the incubation of the isogenic clones of N. gonorrhoeae and S. agalactiae with a medium that was indirectly in contact with the gel containing surfactant and was collected at different time-points. It was observed at 100% efficacy at 24 h and 3-6 h after incubation for N. gonorrhoeae and S. agalactiae, respectively (Fig. 4b).

[0094] Upon incubation with fully polarized Caco-2 cells, both gel formulations demonstrated very low toxicity after each period of C₁₂PB exposure, namely 3 h and 6 h (Fig. 4 c,i). In the same experimental settings and based on the cytokines that are the most common markers found in cervicovaginal wash samples in microbicide studies, minimal inflammation was observed in Caco- 2 medium supernatants after 3 h and 6 h of C₁₂PB-hydrogels incubation, and 24 h after removal of the Ci2PB-containing hydrogel (Fig. 5c, ii-v) (Fig. 4c, ii-v). [0095] Given the similar microbicidal activity and safety profile of both hydrogel formulations, it was selected the F4M-C₁₂PB gel for in vivo studies due to its faster release of C₁₂PB compared to the HEC formulation (Fig. 3c).

IN VIVO STUDIES

[0096] In order to understand the behaviour of C₁₂PB-F4M hydrogel within a living organism, it was performed an in vivo study to evaluate its efficacy, degradation, biodistribution and inflammation profile. The study was carried out on Balb/c mice, with the hydrogel being administered intravaginally, as shown in Fig. 5.

[0097] To track the movement and location of the F4M hydrogel within the body, it was utilized a live imaging system. The hydrogel was labelled with a fluorescent marker, fluorescein isothiocyanate (FITC), to enable its visualization and tracking over a period of 48 hours (Fig. 5a and Sc). The fluorescence from the FITC-labeled hydrogel began to decrease after 6 hours post administration, indicating that the hydrogel was gradually being eliminated from the vaginal tract. However, even after 24 hours post administration, it was observed residual presence of the hydrogel in the vaginal tissue, implying a sustained release or retention of the hydrogel formulation at the site of administration. A particularly significant observation was that the F4M hydrogel primarily accumulated in the vaginal and uterine tract, showing a localized effect of the hydrogel. More importantly, it was found no indication of the hydrogel reaching other off-target organs, which could lead to undesired effects, as displayed in Fig. 5b and d. Comparative analysis of FITC signals across different organs showed significantly lower signals in non-target tissues (liver, spleen, lungs, kidneys, heart) than in the vagina and uterus, indicating that the hydrogel predominantly remains at the site of administration with minimal or residual systemic distribution. This is an important observation, as it suggests a low risk of systemic exposure to the active compound, which is beneficial for safety profiles and is an advantageous property for a microbicide. This represents a reduced risk of systemic side effects and off-target toxicity, making the hydrogel a safer option for the intravaginal delivery of therapeutics.

[0098] These findings are critical for assessing the safety and targeted effectiveness of the C₁₂PB- F4M hydrogel of the present disclosure. It is crucial for the hydrogel to remain localized to the application site and avoid dispersing widely throughout the body. By achieving this, it can be ensured the utmost therapeutic impact at the site of infection while mitigating the risk of potential systemic side effects. Furthermore, the study indicates promising results for applying this hydrogel in sustained or controlled-release applications. This feature allows for prolonged and steady therapeutic action over an extended period, offering enhanced treatment options for various infections.

[0099] It was also assessed the impact of F4M hydrogels containing different concentrations of C₁₂PB on the vaginal tract. Therefore, it was performed an in-depth histological evaluation using the hydrogel comprising different concentrations of C₁₂PB: (10CMC (39000 μM), 5CMC (19500 μM), 1CMC (3900 μM), CMC/10 (390 μM), and CMC/5 (780 μM)). The process involved intravaginal administration of the different F4M hydrogels in BALB/c mice. After a period of 24 hours, the mice were euthanized, and their vaginal tracts were collected for analysis. It was found, as illustrated in Fig. 6, that there was an increased presence of inflammatory infiltrates that is dose-dependent, primarily in the vaginal tissue, with no major observable differences in the uterus and ovaries. It was observed that the hydrogels with the highest concentrations of C₁₂PB, namely the 10CMC and S CMC gels, resulted in mild to moderate inflammation in the vaginal submucosa layer (the tissue beneath the mucous membrane), along with mild edema (swelling caused by fluid accumulation), as seen in Fig. 6b and 6c. The 1CMC hydrogel induced a moderate to minimal inflammatory response in both the submucosa and the muscle layer of the uterus, known as the myometrium. Nonetheless, it was surprisingly found that the hydrogels with C₁₂PB concentrations of CMC/10 and CMC/5) do not provoke significant inflammation, providing results comparable to the control group, which received only the hydrogel without any C₁₂PB. These results were not expected, considering that in vitro results indicate that concentrations above 25 μM (CMC/154) would lead to toxicity.

[00100] It was analysed the presence of CD45 positive cells, which are generally indicative of an immune response. Consistent with the previous histological evaluation, it was found that the hydrogels with higher C₁₂PB concentrations (10CMC, 5CMC, and 1CMC) had a greater number of CD45 positive cells compared to the hydrogels with lower CMC concentrations (CMC/10 and CMC/5), as shown in Fig. 6d. These findings are extremely important to avoid causing genital lesions or inflammation that could potentially increase the risk of HIV infection.¹² These results demonstrate the ability of the topical composition of the present disclosure to minimize adverse inflammatory responses, thus ensuring the safety and effectiveness of the treatment, an essential factor in the development of topical microbicides for preventing sexually transmitted infections.

Microbiome analysis and in vivo antimicrobial evaluation of C₁₂PB in a N. gonorrhoeae preclinical model

[00101] Considering the role of vaginal microbiota in protecting female urogenital health, assessing the impact of the C₁₂PB-hydrogel microbicide of the present disclosure on its composition is of utmost importance. Therefore, upon 24h of intravaginal administration of F4M gel containing C₁₂PB at different CMC (CMC/5 - 780 μM and CMC/10 - 390 μM), BALC/c mice were euthanized, and total DNA was extracted from fresh vaginal tissues for microbiome characterization through 16SrRNA metagenomics (Fig. 7a). It was surprisingly found that the intravaginal application of the C₁₂PB-hydrogel of the present disclosure at concentrations of CMC/5 and CMC/10 only caused slight fluctuations in vaginal microbiota diversity within and between groups of treated mice, likewise seen for the control group (mice treated with hydrogel only). These findings can be seen either by looking at the Shannon entropy, where all samples displayed a similar entropy mean value (2.05-2.23), or by performing a principal component analysis, where samples seem to share similar frequencies of the same taxa, without being clearly separated in the different principal components. This is particularly reassuring as certain taxa within the vaginal microbiome are known to confer protective benefits against pathogens. Our analysis shows that these beneficial bacteria are preserved post-treatment, which is critical for sustaining the natural defence mechanisms of the vagina. These findings collectively indicates that the composition of the vaginal microbiota remains largely unperturbed following the intravaginal application of CMC/5 and CMC/10 C₁₂PB-hydrogel of the present disclosure (Fig. 7b). This observation shows well that the disclosed composition is safe and compatible with the complex vaginal microbial ecosystem, a critical factor in ensuring the overall urogenital health of women. This targeted action is crucial for avoiding dysbiosis, which is often associated with an increased susceptibility to infections and adverse health outcomes.

[00102] Although some AMR strains of GBS have been identified, this situation is particularly critical for N . gonorrhoeae, which is currently resistant to almost all classes of antimicrobials available for treatment. In this circumstance, it was decided to evaluate the antimicrobial efficacy of the C₁₂PB-hydrogel of the present disclosure in a preclinical context only for N. gonorrhoeae. These experiments were conducted in vivo tests using a five-week-old, ovariectomized BALB/c mice. Taking the safety results described above, only CMC/10 and CMC/5 C₁₂PB-hydrogels were tested. In this set of experiments, BALB/c mice were inoculated intravaginally with 9.4x104 CFUs of N. gonorrhoeae (strain FA1090, ATCC 700825). Two hours post-inoculation C₁₂PB-hydrogels were also applied intravaginally. PBS and hydrogel without surfactant were used as control. 1-day post-treatment, mice were euthanized and counts of N. gonorrhoeae were performed in the lavage vaginal fluids (Fig. 7c). From these in vivo results (Fig. 7d) it was found that C₁₂PB-hydrogel at concentrations of CMC/154 (25 μM) and CMC/10 (390 μM) were not effective in reducing N. gonorrhoeae infection. Nonetheless, it was found that a single dose of CMC/5 C₁₂PB-hydrogel significantly reduced N. gonorrhoeae infection when compared with the control groups (mice treated with PBS or with empty-hydrogel). This significant reduction in infection rates, together with the safety and compatibility results, shows that the C₁₂PB-hydrogel of the present disclosure is an innovative, safe and effective intervention strategy against i\i. gonorrhoeae infections, addressing a critical public health concern. The preclinical outcomes from these studies offer key insights into the potential utilization of this hydrogel as a topical treatment for N. gonorrhoeae, showcasing its promise as an effective and safe intervention.

[00103] The topical composition of the present disclosure has the ability to maintain a consistent drug concentration in the vaginal cavity. This is particularly beneficial in a worldwide clinical scenario, as it simplifies the treatment regimen and potentially enhances patient adherence by reducing the need for frequent reapplication. These properties include their ability to absorb large quantities of water and maintain a gel-like consistency, which are both advantageous in providing a comfortable and unobtrusive delivery method. Furthermore, the consistency of hydrogels closely mirrors that of natural biological tissues, which can enhance the user experience and acceptance, particularly in the context of personal applications such as microbicides. In summary, C₁₂PB-hydrogel of the present disclosure represents an innovative approach to combating bacterial infections. It combines in a synergistic way the advantageous properties of the hydrogel herein described, in particular regarding its viscosity, with the potent antibacterial activity of C₁₂PB to provide a safe, effective, and user-friendly alternative to traditional therapies. The low price, chemical stability and non-demanding storage requirements of C₁₂PB makes this compound very attractive for use as a microbicide in topical treatment and prophylaxis of gonorrhoeae. For this reason, the C₁₂PB-hydrogel of the present disclosure proved to have an excellent potential for vaginal drug delivery due to its unique properties.

[00104] The present disclosure shows that C₁₂PB-hydrogel/topical formulation is a potential microbicide to treat N. gonorrhoeae when the available antibiotics fail. This antibiotic-free cationic surfactant-based cellulose hydrogel/topical composition is highly effective, non-disruptive of the integrity of the vaginal epithelial barrier, does not induce mucosal inflammation, interfere with the innate immune response, or alter vaginal flora, and, particularly for the poorer regions of the world, is cheap, easy to store, easy to use, odorless and colorless, and inexpensive enough to allow worldwide distribution. Based on the efficacy of C₁₂PB against GBS in the in vitro studies, it was shown that the C₁₂PB-hydrogel can also be used in perinatal infections.

Methods

[00105] Materials. All mammalian cell culture reagents were purchased from Gibco Life Technologies. N-dodecylpyridinium bromide ( C₁₂PB) was purchased from Sigma-Aldrich. Hydroxyethylcellulose (HEC, natrosol 250 HX pharm) was kindly provided by (Ashland, Safic-Alcan Especialidades, S. A. U., Spain) and hydroxypropylmethylcellulose (Methocel F4M) was kindly provided by (Colorcon Limited, UK). Commercially available reagents were used without further purification and all solvents were of HPLC quality, purchased from Sigma-Aldrich, Fluorochem, Alfa Aesar, TCI, Carlo Erba or Honeywell.

[00106] Hydrogel-FITC chemical modification. HPMC (2g, containing 4.0 - 7.5% Hydroxypropoxyl) was dissolved in 20mL DMF over lh. Triethylamine (0.77 mM, 107 μL) and FITC (100 mg, 0.26 mM) were added to the solution and the reaction was stirred overnight at 80°C under nitrogen atmosphere. The HPMC was precipitated out of reaction mixture into 200 mL diethyl ether, washed with ethanol (2x 100 mL) and then dried under vacuum. The solid was dissolved in water and dialyzed (1 kDa cut-off) for 5 days against water, past which time was lyophilized to yield HPMC-FITC.

[00107] Reactions under the N2 atmosphere were performed in ovendried glassware and anhydrous solvents. Anhydrous DCM and THF were obtained using a Pure Solv™ Micro 100 Liter solvent purification system with activated alumina column. All reactions were monitored by thin layer chromatography using Merck silica gel 60F254 aluminum plates. Visualization of the former was conducted under UV-light. Flash chromatography was performed with Merck Geduran® Si 60 (0.040-0.063 mm) silica gel. NMR spectra were recorded in Bruker Fourier 300 using CDCI3. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project N- 022161c and ROTEIRO/0031/2013- PINFRA/22161/2016 (financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). All coupling constants are expressed in Hz and chemical shifts (5) in ppm. Multiplicities are given as: s (singlet), d (doublet), dd (double doublet), dt (double triplet), t (triplet), td (triple triplet), tt (triple triplet), q (quartet), quint (quintuplet) and m (multiplet).

[00108] Collected supernatants analysis by LC-MS. The Liquid chromatography- mass spectrometry (LC-MS) runs were performed using a Dionex Ultimate 3000 UHPLC+ system equipped with a Multiple-Wavelength detector and an imChem Surf C18 Tri F 100 A 3 pm 100 x 2,1 mm column connected to Thermo Scientific Q Exactive hybrid quadrupole Orbitrap mass spectrometer (Thermo ScientificTM Q ExactiveTM Plus), Figure 12.

[00109] Cell culture. The human intestinal columnar epithelial cell line Caco-2 (ATCC® HTB-37™) was obtained from the American Type Culture Collection (ATCC) and cultured in complete growth medium that consists of Dulbecco's Modified Eagle Medium with GlutaMAX™, supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin-streptomycin (P/S), 1 mM of sodium pyruvate, and 1 mM of non-essential amino acids. Cells were grown in a humidified incubator at 37°C under 5% CO₂. 10 days after seeding, Caco-2 cells were incubated with C₁₂PB or Reduced Serum Medium (Opti-MEM™) as control as indicated in the figure legends.

[00110] Bacterial Strains, Characterization and Growth. In this study, two clinical isolates of i\i. gonorrhoeae and S. agalactiae belonging to the stock culture collection of the STI Reference Laboratory from the Portuguese National Institute of Health were evaluated. All experiments were performed using isogenic clones of both isolates previously produced. For N. gonorrhoeae, the antimicrobial susceptibility testing against azithromycin, benzylpenicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, spectinomycin and tetracycline was determined by E-test® (bioMerieux) on Chocolate agar PolyVitex (bioMerieux), as previously described.¹³ The production of P-lactamase was also tested using the chromogenic reagent nitrocefin (Oxoid), according to the manufacturer's instructions. For 5. agalactiae, antimicrobial susceptibility was examined with the automated VITEK®2 AST-P586 card (bioMerieux), which evaluates a panel of 13 antibiotics (amoxicillin, benzylpenicillin, clindamycin, erythromycin, moxifloxacin, levofloxacin, linezolid, nitrofurantoin, teicoplanin, tetracycline, tigecycline, trimethoprim-sulfamethoxazole and vancomycin), according to the manufacturer's instructions. For both gonococcal and streptococcal isolates, antibiotic resistance was classified according to the current European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint definitions (www.eucast.org/clinical_breakpoints). For each isolate, the C₁₂PB (Sigma-Aldrich) minimum inhibitory concentration (MIC) was assessed by the broth microdilution method ¹⁴, as previously described.⁸ Briefly, an inoculum density of 5x10⁵ CFU/ml, growth media and incubation conditions were performed according to EUCAST guidelines.¹⁴ N. gonorrhoeae culture was aerated by shaking. The MIC was determined as the lowest C₁₂PB concentration at which no visible growth was observed. Bacterial growth curves were generated in 24-well plates in the absence and presence of C₁₂PB at sub-MIC (1/2 , 1/4, 1/8), MIC and 2xMIC conditions in appropriate medium. Inoculum was prepared according to EUCAST guidelines⁷⁷ in order to ensure a final concentration of 5x10^s CFU/ml per well. Negative (broth) and positive (broth with bacteria) controls were also performed. Bacterial growth rate was evaluated during 20-50 h by ODBOOnm measurements using the spectrophotometer GeneQuant Pro (GE Healthcare / Amersham Biosciences). The assay was performed in duplicate.

[00111] In vitro selective pressure assay. For each bacterial strain, independent in vitro C₁₂PB selective pressure assays were performed under continuous sub-MIC conditions for creating C₁₂PB non-susceptible strains (Meinike, J. K.; Tina, W.; 0strup, J. P.; Wang, H.; Sοren, M.; Niels, H.; Oana, C. Sublethal Ciprofloxacin Treatment Leads to Rapid Development of High-Level Ciprofloxacin Resistance during Long-Term Experimental Evolution of Pseudomonas Aeruginosa. Antimicrob. Agents Chemother. 2013, 57 (9), 4215-4221, httffs^c^ J Gullberg,

E.; Cao, S.; Berg, O. G.; Ilback, C.; Sandegren, L.; Hughes, D.; Andersson, D. I. Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLOS Pathog. 2011, 7 (7), el002158-)

[00112] Briefly, isogenic clones of the selected strains (5xl0⁵ CFU/ml) were continuously subcultured on a 24-well plate, at the exponential growth phase, in appropriate medium [Fastidious broth medium (FB), prepared as described by Catwright et al. for N. gonorrhoeae, ¹⁵ and Todd- Hewitt broth (Difco) for S. agalactiae], under the following conditions: I) with a constant C₁₂PB sub-MIC (% MIC for N. gonorrhoeae and 1/4 MIC for S. agalactiae, eight replicates each) ensuring a proportion of 1% of compound per well; and ii) without C₁₂PB (positive control, four replicates) to discard mutations that may emerge due to laboratory passages. Negative controls (four replicates) were also added to check for possible contaminations occurring in the daily preparation of each plate or during the successive passages. After incubation at 37°C and 5% CO₂ during ~24 h with shaking for N. gonorrhoeae and ~12 h for S. agalactiae, plates were continuously passed. Prior to each passage, ODeoonm was measured to assess bacterial growth and to determine the amount to transfer from each well to a new microplate, without creating any bottleneck. The emergence of resistant clones to C₁₂PB was phenotypically evaluated every six to seven passages, by testing a sample of each well at different C₁₂PB concentrations in GC agar plates supplemented with 1% isovitalex (BD BBL™) and in Todd-Hewitt broth medium for N. gonorrhoeae and S. agalactiae, respectively. Throughout the assay, wells content of all evolved bacterial populations was collected for culture preservation at -80°C in 25% glycerol as well as for DNA extraction and posterior whole genome sequencing (WGS). At the end of the N. gonorrhoeae selective pressure assay, to assess the reproducibility of the obtained data, which showed a 1-fold MIC increase (see results for details), it was performed a parallel experience by restarting the whole C₁₂PB selective pressure assay from passage 25 (the latest without phenotypic changes) (Fig. 3a). Briefly, 200μL of each gonococcal population frozen at P25 (four replicates without and eight replicates under C₁₂PB exposure) were placed in culture on a 24-well plate containing FB medium, under the same conditions described above, being continuously propagated every 24 h for more 80 extra passages. As a proof-of-concept, selective pressure tests were also carried out in sub-MIC conditions on the same isogenic clones with azithromycin (AZT) and erythromycin (ERY) (Sigma-Aldrich), which are antibiotics previously recommended in the prophylaxis of N. gonorrhoeae and S. agalactiae, respectively, and are known to induce antimicrobial resistance in these microorganisms. Therefore, for each antibiotic, bacterial growth curves were also obtained in the same conditions described above. For N. gonorrhoeae, phenotypic susceptibility evaluation was performed by determining the MIC with ETEST® (bioMerieux) as described above. On the other hand, for S. agalactiae, susceptibility to ERY was initially performed in Todd Hewitt broth medium, being replaced by ETEST® (bioMerieux) on Columbia agar plus 5% sheep blood agar (bioMerieux) when MICs exceeded 16-fold the baseline value. MIC values obtained are the median values of two ETESTs from biological replicates and were interpreted according to the breakpoints stated by EUCAST (www.eucast.org/clinical breakpoints). For both N. gonorrhoeae and S. agalactiae, in order to check for colony purity and discard possible contaminations during the in vitro selective pressure assay with C₁₂PB, AZT and ERY, the content of randomly selected wells was frequently analyzed by MALDI-TOF (VITEK® MS system, bioMerieux).

[00113] Evaluation of antibiotic cross-resistance. In order to evaluate whether C₁₂PB may enhance antibiotic resistance, 10 μL of all gonococcal and streptococcal populations from the latest continuous passage under surfactant sub-lethal concentration were placed in culture on appropriate C₁₂PB-free agar plates. After incubation at 37°C and 5% CO₂ during 24 h, each bacterial population was examined regarding its antimicrobial susceptibility against the panel of antibiotics previously tested, as described above. Antibiotic resistance was classified based on current EUCAST breakpoints. For N. gonorrhoeae, the production of 0-lactamase was also tested using the chromogenic reagent nitrocefin. These results were then compared to those obtained initially for the respective original isogenic clone.

[00114] Whole Genome Sequencing (WGS). Considering the phenotypic results obtained for the replicates completely followed until the end of each assay, besides the two original clones, 84 gonococcal and 69 streptococcal populations from well suspensions from different passages were selected for WGS (Figure 8), in order to ensure a robust representative sampling. Genomic DNA was extracted from bacterial suspensions using the Isolate II genomic DNA kit (Bioline) following the manufacturer's instructions and quantified in Qubit® fluorometer 3.0 (Invitrogen) using the QubitTM dsDNA HS Assay Kit (Invitrogen). DNA samples were then subjected to the Nextera XT Illumina library preparation protocol (Illumina), prior to paired-end sequencing (2xl50bp for N. gonorrhoeae or 2x250bp for S. agalactiae) on an Illumina MiSeq, NextSeq550 or NextSeq 2000 equipment (Illumina) available at the Portuguese NIH, according to the manufacturer's instructions. Reads quality control and improvement, species confirmation (using the 8GB database available at https://ccb.jhu.edu/software/kraken/) and bacterial de novo assembly were performed using the INNUca v4.2.2 pipeline (https://github.com/B-UMMI/INNUca).¹⁶ MLST prediction was determined using mist V2.18.1 software (https://github.com/tseemann/mlst). All gonococcal assemblies were further in silica typed with NG-MAST v2.0, upon query to the PubMLST Neisseria database (http://pubmlst.org/neisseria/). Draft genome sequences (n=85 for N. gonorrhoeae and n=70 for S . agalactiae) were annotated with RAST server v2.0 (http://rast.nmpdr.org/).¹⁷ For each tested compound, the identification of putative mutations and/or fluctuations in accessory genome that may be responsible for AMR was performed through comparative whole genome analysis between isogenic clones propagated under selective pressure versus the original isogenic clone ("ancestral"). In order to inspect the presence/absence of accessory genome, N. gonorrhoeae and S. agalactiae assemblies were aligned using the progressive algorithm of MAUVE v2.3.1 software (http://darlinglab.org/mauve/mauve.html).¹⁸ Moreover, assemblies were also analyzed using PHASTER (http://phaster.ca/)¹⁹ to assess the presence of prophages, the MobileElementFinder

(https://cge.food.dtu.dk/services/MobileElementFinder/)²⁰ to detect mobile genetic elements (MGEs) as well as the PubMLST Neisseria and S. agalactiae databases (http://pubmlst.org/neisseria) to examine the presence of the gonococcal genetic island and of the conjugative, P-lactamase and cryptic plasmids for N. gonorrhoeae, or specific S. agalactiae MGEs (ICESagl962 and IMESp2907). Additionally, the existence of putative plasmids for S. agalactiae was inspected with PlasmidFinder 2.1 (http://cge.cbs.dtu.dk/services/PlasmidFinder/) using default parameters. On the other hand, Snippy v4.5.1 software (https://github.com/tseemann/snippy) was used to disclose genomic markers for different levels of susceptibility to each tested compound as well as to identify the intrinsic putative resistance mechanisms. Basically, quality improved reads of the evolved populations under C₁₂PB, AZT or ERY pressure were individually mapped against the draft genome of the original isogenic clone of N. gonorrhoeae or S. agalactiae. Variants were called on sites that filled the following criteria: I) minimum mapping quality and minimum base quality of 20; ii) minimum number of reads covering the variant position >10; and ill) minimum proportion of reads differing from the reference of 90%. For both N. gonorrhoeae and S. agalactiae, after discard all variants arose from laboratory passaging, all single nucleotide variants (SNVs) and indels acquired under C₁₂PB, AZT and ERY pressure were carefully inspected and confirmed using IGV V2.12.3 (http://software.broadinstitute.org/software/igv/).¹⁴ In parallel, in silico identification of AMR determinants was also performed by querying sequences against the ResFinder 4.1 (https://cge.food.dtu.dk/services/ResFinder/)²¹ and the Comprehensive Antibiotic Resistance Database 3.2.5 (CARD - RGI 6.0.0) (https://card.mcmaster.ca/analyze/rgi)²² web servers (accessed in November 2022). For all gonococcal populations, in silico AMR was further determined with the NG database of ARIBA V2.14.6 software.²³ For interpretation purposes, typing with NG-STAR⁶⁹ and PubMLST 5. agalactiae database was performed. The predicted results were then compared with antimicrobial susceptibility testing results.

[00115] Data availability. Raw sequencing reads of the sequenced N. gonorrhoeae and S. agalactiae populations were deposited in the European Nucleotide Archive (ENA) under the BioProject accession number PRJEB66433.

[00116] Hydrogel preparation. Hydroxyethylcellulose at 1.25 wt. % (HEC, Natrosol (Ashland) and hydroxypropylmethylcellulose at 2 wt. % (Methocel F4M, Colorcon) were prepared in PBS or sterilized water for in vivo studies. C₁₂PB surfactant was added to the aqueous solution and homogenized for 2 min at 16 000 rpm using Ultra-Turrax equipment (IKA, Germany). Then, the solution was sterilized under UV light for 15 minutes and stored at 4°C until further use.

[00117] Rheology studies. Cellulose-based hydrogels were performed as previously described, containing 25 μM of C₁₂PB. An MCR 501 rheometer (Anton Paar, Austria) with a hood for temperature control (37 °C) was used to measure the viscoelastic properties of cellulose-based hydrogels. In shear sweep tests a 25 mm plate/plate geometry was used. The solution's viscosity was measured in response to the increasing shear rate from 0.01 to 1000 s-1. In strain frequencysweep tests, hydrogels were formulated as described above. At a fixed strain amplitude value within the linear viscoelastic region (3%), selected within the linear viscoelastic region, frequency sweeps from 0.01 to 100 Hz were performed to obtain the frequency dependence of the storage modulus (G') and loss modulus (G"). All measurements were performed in triplicate.

[00118] Hydrogel microstructure. After casting into the molds, hydrogel containing 25 μM of C₁₂PB was frozen at -80 °C and subsequently freeze-dried overnight. Moreover, the samples were freeze-fractured, after immersion in liquid nitrogen, to expose their inner structures and sputter coated (60 seconds at 20 mA, Cressington) with gold prior to observation in a scanning electron microscope (JSM-6010LV, JEOL, Japan).

[00119] Cumulative profile release of surfactant from hydrogel. Transwell cell culture inserts with 0.4 pm pore membrane (Corning, USA) were used to study the profile release of surfactant from the hydrogel. Cellulose-based hydrogels were prepared as previously described containing 50 μM C₁₂PB surfactant. Then, 500 μL of cellulose-based gel was deposited in the upper part of the Transwell membrane, and S00 μL of miliQ water was added to the well, and fresh PBS was added to the well. At different time points, the supernatants were collected and kept at 4° C until further analysis. The collected supernatants were monitored at 0, 20, 60, 120, 150, 360 and 1440 min by LC-HRMS in Positive Mode. The HPLC runs were carried out with a gradient of A (Milli Q water containing 0.1% v/v Formic acid, FA) and B (acetonitrile containing 0.1% v/v FA, Honeywell HPLC-grade). The mobile phase was t=0 min, 1% B; t=15 min, 99% B; t=17 min, 95% B; t=18 min, 1% B; t=20 min stop, at a flow rate of 0.3 mL/min. Quantification was performed according to EIC intensity (AUC) within the δ 6ppm range.

[00120] In vitro toxicity evaluation of C₁₂PB and C₁₂PB-hydrogels towards polarized Caco-2 cells. For viability assays, Caco-2 cells were plated at an initial density of 32000 cells/ well in 96-well plate. The cells were allowed to grow until they became fully polarized (~10 days). Cells were then incubated with different concentrations of C₁₂PB (from 0.65 μM to 3900 μM) prepared in Opti- MEM™ for 20, 60, 180 and 360 min. For C₁₂PB-Hydrogel procedures, cells were incubated with both C₁₂PB formulations (HEC and F4M) prepared in Reduced Serum Medium (Opti-MEM™) for 3 h and 6 h. After each incubation, the wells were replaced with fresh complete DMEM media without phenol red. Cells were kept for more ~24 h in culture, and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, as previously described.⁷ Briefly, cells were incubated with MTT reagent (final concentration of lmg/ml) in cell culture medium without phenol red for 2 hours at 37°C, 5% CO2 followed by the addition of the solubilization solution (16% SDS in 50% DMSO) overnight. Samples were quantified colorimetrically at 570 nm (background wavelength correction at 650 nm) on a SpectraMax i3x microplate spectrophotometer (Molecular Devices). Background absorbance (culture medium plus MTT without cells) was subtracted from the absorbance of each sample and data are shown as a percentage of control (untreated cells). All cell cultures and incubations were performed at 37°C in 5% CO2. All assays were done in triplicate.

[00121] Quantification of inflammation by the Enzyme-Linked Immunosorbent Assay (ELISA). Quantification of a pool of pro-inflammatory human cytokines and chemokines commonly used as biomarkers of inflammation in vaginal microbicide studies¹¹ was performed in Caco-2 supernatants by ELISA, accordingly to the manufacturer's instructions (Human uncoated ELISA kits, Thermofisher Scientific). For that purpose, Caco-2 cells were incubated with sub-toxic concentrations of C₁₂PB (3.9 μM and 25 μM), corresponding to the MIC range of several clinical isolates of N. gonorrhoeae and S. agalactiae for 20, 60, 180 and 360 min.⁸ For the C₁₂PB-hydrogel experiments, only IL-6 and IL-8 were assessed in Caco-2 supernatants exposed for 3h and 6h to the surfactant-containing hydrogels. Cell culture media was collected immediately after incubation or 24 h after formulation exposure and clarified at 1200 rpm, 5 min, 4 °C to remove cell debris and stored at -80 °C until further use. NuncTM MaxisorpTM 96-well plates were coated with anti- IL-8, anti-IL-6, anti-TNF-a, anti-IFN-y, anti-IL-18, anti-MCP-l/CCL2, anti-MIP-l/CCL3 and anti-IL-10 antibodies and incubated at 4°C overnight. Plates were then washed with PBS 0.05% tween-20 and blocking was performed with ELISA/ELISPOT Diluent (lx). After washing, samples and cytokine/chemokine standards were added in appropriate serial dilutions, and plates were incubated at 2 h at room temperature (RT). Samples were not diluted. Plates were incubated with the respective detection antibodies for 1 h at RT, and streptavidin-conjugated horseradish peroxidase was added for 30 min. Tetramethylbenzidine (TMB) was added followed by the addition of H3PO4 1 M (stop solution) after 15 min at RT. OD values were measured at 450 nm on a SpectraMax i3x microplate spectrophotometer (Molecular Devices), and absolute concentration of cytokines and chemokines (pg/ml) was obtained from their respective standard curves. Cells treated with LPS (10 pg/ml) and/or 40 ng/ml of IL-1β, TNF-a and IFN-y (alone or in combination) for 24 h in complete DMEM medium were used as positive controls. Cytokine and chemokine levels were measured in triplicate.

[00122] Antibacterial activity of C₁₂PB-hydrogels. C₁₂PB-hydrogel efficacy was evaluated by assessing C₁₂PB-hydrogels antibacterial activity against i\i. gonorrhoeae and S. agalactiae isogenic clones by direct and indirect assay for several incubation conditions. In the direct assay procedure, equal volumes (1:1) of each hydrogel formulation (HEC and F4M) containing 50 pm C₁₂PB surfactant plus the appropriate culture medium were transferred to a 96-well plate. A bacterial inoculum of N. gonorrhoeae and S. agalactiae isogenic clones was prepared according to EUCAST guidelines, as previously described, and was added to the respective wells in order to achieve a final concentration of 5xl0⁵ CFU/ml per well. Plates were incubated for 20 min, 60 min, 2 h, 3 h, 6 h, 8 h, 10 h, 12 h, 16 h, 18 h and 24 h at 35-37°C, 5% CO₂. In the indirect assay procedure, transwell cell culture inserts with 0.4 pm pore membrane (Corning, USA) were used to study the profile release of surfactant from the hydrogel. This indirect assay method for evaluating microbicide efficacy is important, as it simulates the conditions of actual product use more closely, where the microbicide may not directly contact pathogens. This method complements direct assay results and provides a more holistic evaluation of the efficacy of microbicides. Cellulose-based hydrogels containing 50 pm C₁₂PB surfactant were prepared as previously described. Then, 500 μL of cellulose-based gel was deposited in the upper part of the Transwell membrane, and S 00 μL of bacterial completed medium was added to the well. At different time points (0, 20, 60, 120, 150, 360 and 1440 min), the supernatants were collected and kept at 4° C until further analysis, and fresh medium was added to the well. The collected supernatants were transferred to a 96-well plate and a bacterial inoculum of N. gonorrhoeae and S. agalactiae isogenic clones (prepared as previously described) was added to the respective wells achieving a final concentration of 5xl0⁵ CFU/ml per well. Plates were incubated for 24h at 35- 37°C, 5% CO₂. Negative (broth and Hydrogel without the surfactant) and positive (broth with bacteria) controls were also performed. For comparative purposes, bacterial growth was also evaluated under the exposure of only C₁₂PB. At the end of each incubation, the content of each well was serially diluted (10-¹, 10 ², 10³, 10⁴) and 100 pl of each dilution was spread onto agar plates (chocolate agar for N. gonorrhoeae and with 5% Sheep Blood for S. agalactiae) and incubated for more 24 h under the appropriate conditions. After each incubation, visible colonies were counted and the number of CFUs were calculated for each condition ([number of colonies/y (plated volume x dilution factor)]. The C₁₂PB-hydrogels antibacterial efficacy was calculated by normalizing the number of CFUs/ml obtained for condition/incubation time to the number of CFUs/ml in the positive control of infection (hydrogel without C₁₂PB) for the respective condition. Results were obtained from three independent assays.

[00123] BALB/c mice animal model. Female Balb/cByJ (6-8 weeks old) mice were purchased from Charles River Laboratories and maintained according to the protocols approved by the national (Portuguese Official Veterinary Department; Diregao Geral de Veterinaria) ethics committees according to the Portuguese (Decreto-Lei 113/2013) and European (Directive 2010/63/EU) legislations. After acclimatization, mice were injected subcutaneously with 2.5mg of medroxyprogesterone acetate (Depo-Provera; Pfizer, NY, USA) in PBS. The further procedures were performed one week after the Depo-Provera administration.

[00124] In vivo tolerance to cationic C₁₂PB-hydrogel formulations. The mice were randomized into 6 groups. Animals received a single treatment of 50 μL of F4M C₁₂PB (Group 1, n=3, 39000 μM C₁₂PB), F4M C₁₂PB (Group 2, n=3, 19500 μM C₁₂PB), F4M C₁₂PB (Group 3, n=3, 3900 μM C₁₂PB), F4M C,₂PB (Group 4, n=3, 780 μM C₁₂PB), F4M C₁₂PB (Group 5, n=3, 390 μM C₁₂PB), F4M hydrogel only (Group 6 n=4) by intravaginal application with a soft-tipped plastic tube 16ga/38 mm. Mice were euthanized the following day by cervical dislocation, and the organs were collected for histology. The mice were euthanized, and the organs were collected the day after the last intravaginal treatment. All intravaginal administrations were performed under isoflurane anesthesia (IsoFlo 100% p/p).

[00125] Fluorescent-labeled hydrogel biodistribution in vivo. Four mice received a single treatment of 50 μL of fluorescent-hydrogel by intravaginal application with a soft-tipped plastic tube 16ga/38mm. After receiving the treatment, fluorescence imaging was performed to each group using the Newton FT500 imaging system (Vilber) after excitation at 440 nm. Fluorescence imaging procedures were repeated at 20 min, 1 hour, 3 hours, 6 hours and 1, 2, and 3 days after the injection. At the endpoint (day 3), mice were euthanized by cervical dislocation. The vaginal tract was collected and imaged for fluorescence analysis.

[00126] Histological procedure and evaluation. The presence of epithelial damage in mouse vaginas, cervix, uterus, and ovaries was accessed by hematoxylin-eosin staining. Briefly, formalin- fixed paraffin embedded (FFPE) samples were sectioned at 3 pm thickness, mounted on positively charged glass slides and dried overnight at 65°C. After deparaffinization, samples were rehydrated and stained with haematoxylin and eosin (H&E). H&E staining was evaluated by a histological score, which is expressed in ordinal scale units. Staining intensities of IHC from vaginal tissue per treatment group were determined using ImageJ software. Immuno-staining was performed with the automated immunohistochemistry (IHC) Autostainer Link 48 (Agilent). First, sections were submitted to low pH heat-induced epitope retrieval with EnVision FLEX Target Retrieval Solution, Low pH (K8005, Agilent) for 20 minutes. Peroxidase-Blocking Reagent (K800, DAKO/Agilent) was used for 30 minutes to block endogenous peroxidase activity. Sections were then incubated with 1% bovine serum albumin (biowest, USA) for 1 hour. Afterwards, staining was performed using EnVision FLEX Visualization System protocol. Primary antibody incubation time was overnight. Tumor sections were stained for CD45 (abl0558). The secondary antibody combined with HRP was added for 1 hour. Immunohistochemistry evaluation was determined by CD45 positive Cell Detection. Briefly, files were loaded onto a project in QuPath software (QuPath source code, documentation, links to the software download are available at For IHC analysis, the image type was changed from H&E to DAB to reflect the chromogen used for immunostaining. A representative region of interest of the Vagina was selected with the polygon tool. Stains were digitally separated using the colour deconvolution method and the automated "Estimate stain vectors" function in QuPath. Watershed cell nucleus detection was performed and optimized visually using the following settings: Hematoxylin (OD); requested pixel size 0.5 pm; background radius 8.0 pm; median filter radius 0 pm; sigma 1.5 pm; min/max area 10/400 pm; threshold 0.1; maximum background intensity 2.0; and cell expansion 1,037 pm. The single threshold value for CD45-positivity was selected. The results are expressed as the number of positive cells per area um². Grading system for microscopic examination of vaginal tissue reaction (ISO 10993-10) with scores: 0 (no change) when no injury or the observed changes were within normal range; 1 (minimum) when changes were sparse but exceed those considered normal; 2 (light) when injuries were identifiable but with no severity; (3) moderate for significant injury that could increase severity; 4 (very serious) for very serious injuries that occupied most of the analyzed tissue. The score was used to evaluate the epithelial integrity, epithelial vascular congestion, inflammatory infiltrate, and edema fibrosis of each formulation. These values were summed to obtain a total score and determine the overall histological health of the vaginal tract tissues as minimum 1-4; average 5-8; moderate 9-11 and severe 12-16. Thus, the total score reflects the cumulative effect of the hydrogel on the tissue structure and immune response, offering insights into its potential for clinical use and the need for optimization to minimize tissue disruption and inflammation.

[00127] In vivo vaginal microbiome characterization. After the in vivo experiment, mice were euthanized by cervical dislocation. The vaginal tract was collected and then cryopreserved under liquid nitrogen and kept at - 80°C until further analysis. Isolated DNA from all samples were quantified using Qubit^HS kit (Invitrogen, Life Technologies), and subsequently diluted, accordingly, in order to achieve the required 5 ng/pl concentration. All DNAs were then used to prepare 16S Ribosomal RNA Gene amplicons for metagenomic sequencing by the Illumina MiSeq System, according to Illumina specifications ("16S Metagenomic Sequencing Library Preparation" procedure, Part # 15044223 Rev. B, Agilent Technologies). Briefly, 2.5 pl of each DNA (5 ng/pl) were amplified using 5 pl of each primer (16S Amplicon PCR Forward Primer: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG (Seq. ID 1); 16S Amplicon PCR Reverse Primer: 5'-GTCTCGTGGGCTCGGAGATGTGTATAA

GAGACAGGACTACHVGGGTATCTAATCC) (Seq. ID. 2) (Table 18) and 12.5 pl of 2x KAPA HiFi HotStart ReadyMix (Roche Diagnostics), in a final reaction volume of 25 pl.

Table 18. The following table describes the list of sequences described in the present application, for which an accession number is not available.

[00128] Amplification conditions were as follows: 95°C for 3 minutes, 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, 72°C for 5 minutes, and a final hold at 4°C. PCR products were run on a Fragment Analyzer instrument (Agilent Technologies) to estimate the sample concentration. Because of the very low concentration of amplicons, indexing PCR was performed using 15 pl (instead of 5 pl) of DNA and the library products were vacuum dried to a final ~10 pl volume after the second clean step. The libraries were run on the Fragment Analyzer to confirm the expected product sizes and estimate their concentration. Libraries were normalized to a concentration of 0,1 nM using sterile distilled water and pooled using 4 pl each. The library pool was concentrated ~20X by vacuum drying and subsequently quantified using the Qubit® fluorometer 3.0 and the QubitTM dsDNA HS Assay Kit (Invitrogen). The pool was denatured and diluted to a final concentration of 6 μM. The loading library pool contained 25% PhiX at a concentration of 6 μM. Sequencing was performed in the MiSeq system (Illumina) using 2x300 bp reads. All 16S rRNA data were analyzed with Kraken V2-2.1.2 with default parameters, matching against the Greengenes 16S rRNA database (version 13.5). Within each sample, it was calculated the relative frequency of taxa merged at the family level, to promote a balance between reliability and resolution. With these frequencies, it was calculated the Shannon entropy and performed a principal component analysis. Moreover, based on the preliminary pre-clinical in vivo efficacy assays to treat N. gonorrhoeae infection (see results for details), it was also assessed which C₁₂PB concentration should be loaded into hydrogel. Therefore, mice were randomized in 3 groups (9 animals, 2-4 per group). Group 3 received a single treatment of 50 μL of F4M hydrogel only, while F4M hydrogel with two different concentrations of C₁₂PB were used for the remaining groups (group 1: CMC/5; group 2: CMC/10), by intravaginal application with a soft-tipped plastic tube 16ga/38mm. Mice were euthanized on the following day by cervical dislocation and fresh vaginal tissues were collected for total DNA extraction. Tissue samples were sliced into small fragments and macerated in PBS with a pestle until no clumps were visible. Samples were then transferred into a Pathogen Lysis Tube L and subjected to homogenization on a FastPrep-24 instrument (6.5 m/s for 45 s, twice, with a 5-min interval). DNA isolation proceeded using the QIAamp’ DNA Mini Kit according to manufacturer's instructions ("Tissues" section, QIAGEN). Preparation of 16S rRNA gene amplicons, sequencing and bioinformatic analysis were performed as described above.

Preclinical anti-bacterial C₁₂PB-hydrogel assessment against gonococcal infection

[00129] To test pre-clinical in vivo antimicrobial evaluation, a Neisseria gonorrhoeae ATCC 700825, mouse vaginal infection model was used to assess the antimicrobial efficacy of C₁₂PB-hydrogels for treatment of female gonorrhea. Briefly, groups of 5 ovariectomized BALB/c mice 5 weeks of age are used. Animals were subcutaneously injected with a water-soluble form of estradiol at 0.23 mg/mouse on day -2, 0, 2, 4 and 6. Starting from day -2 and following inoculation to the study end, mice are dosed twice daily with streptomycin (1.2 mg/mouse) and vancomycin (0.6 mg/mouse) by IP injection to minimize the vaginal flora. Trimethoprim sulfate 0.04 g/100 mL is administered in drinking water. At day 0, mice are anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg) through IP injection, and then inoculated intravaginally with 9.4xl0⁴ CFU of N. gonorrhoeae (FA1090, ATCC 700825). Before inoculation, the mouse vagina is first rinsed with 30 μL of 50 mM Hepes (pH 7.4) followed by inoculation with 20 μL gonococci suspension in PBS containing 0.5 mM CaCI₂ and 1 mM MgCI₂. Hydrogels containing C₁₂PB are administered two hours after inoculation. Mice are euthanized with CO₂ asphyxiation at 1-day post-hydrogel intravaginal application. Vaginal lavage was performed with 400 μL GC broth containing 0.05% saponin to recover vaginal bacteria. The bacterial suspensions were plated onto chocolate agar to determine the N. gonorrhoeae counts.

Initial N. gonorrhoeae and S. agalactiae clone characterization

[00130] Antimicrobial susceptibility testing (AST) (Table 6) showed that the N. gonorrhoeae isolate is susceptible to AZT, ciprofloxacin, spectinomycin, ceftriaxone and cefixime. It also displays reduced susceptibility to benzylpenicillin (type II non-mosaic allele 2.001) and gentamicin, as well as low-level resistance to tetracycline due to the amino acid alteration V57M in ribosomal protein S10 RpsJ.^26,27 Although susceptibility was not tested, in silico prediction based on Whole Genome Sequencing (WGS) data from isolates revealed that it also possess the H552N and R228S mutations in the RNA polymerase subunit B (RpoB) and dihydropteroate synthase FolP, implicated in rifampicin and sulphonamides resistance, respectively.²⁸³⁰ Moreover, the A39T/R44H double mutation was found in the MtrR transcriptional repressor of the MtrCDE efflux pump, which is associated with decreased susceptibility to hydrophobic antimicrobials like macrolides, β-lactams antimicrobials, ciprofloxacin, and tetracycline.^29,31 Regarding the S. agalactiae colonizing strain, susceptibility was observed for 12 out of the 13 antibiotics tested (Table_7_GBS); the exception occurred for tetracycline, for which the observed phenotypic resistance is due to the presence of tetM gene carried by a Tn916 transposon.³²³³ Moreover, for the selected antibiotic (ERY), determination of the minimal inhibitory concentration (MIC) revealed values of 0.125 pg/mL (using Todd Hewitt broth) or 0.19 pg/mL (using Columbia agar + 5% sheep blood). Regarding C₁₂PB, MICs of 25 μM and 12.5 μM were determined for N. gonorrhoeae and S . agalactiae isolates, respectively, and were considered as baseline values for the subsequent assays.

^[00131] Finally, since horizontal gene transfer may be an important adaptive path to antibiotic resistance,³⁴ both clinical isolates were roughly inspected regarding its accessory genome. Nevertheless, no intact prophages were found for both isolates nor plasmids and known mobile genetic elements (IMESp2907 and ICESagl962) for S. agalactiae. The N. gonorrhoeae isolate was found to carry the cryptic plasmid (pST-3), but not the conjugative or p-lactamase plasmids nor the gonococcal genetic island, a type 4 secretion system that was significantly associated with AMR to multiple antimicrobials. ³⁵

Growth curves

[00132] In order to set down the conditions of the subsequent in vitro selective pressure assays, bacterial growth behavior was inspected. For N. gonorrhoeae, it is well established that at 24 h bacteria are still exponentially growing in FB medium, even in the presence of different sub-MIC conditions of various antibiotics.³⁶³⁹ Thus, % MIC was chosen to carry out the gonococcal selective pressure assays with continuous passages performed every 24h.

[00133] Contrarily to N. gonorrhoeae, for the also fastidious S. agalactiae, literature regarding bacterial growth is scarce and not so well supported. Considering that S. agalactiae is a faster growing bacterium when compared to N. gonorrhoeae, streptococcal growth curves were performed in the absence and under different concentrations of C₁₂PB and ERY (Figure 11). Interestingly, C₁₂PB sub-MICs seem not to affect S. agalactiae growth with almost no differences observed in the growth behaviour between C₁₂PB sub-MIC exposure (14, %, %) and non-exposure (positive control). On the other hand, for ERY, while no significant differences were seen for % MIC and 1/4 MIC, a different scenario was found for % MIC, where bacterial growth was clearly affected, with S. agalactiae clone achieving lower cell densities and exhibiting an extended lag phase (~7-8 h). For both compounds, an exponential phase of about 10-12 h was observed and as expected, no growth was observed at the MIC. Therefore, 1/4 MIC was chosen to perform the subsequent S. agalactiae pressure assays (as it not significantly impacts bacterial growth) with passages occurring every 12 h.

[00134] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[00135] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[00136] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[00137] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[00138] As used herein, the term "about" a number refers to that number plus or minus 10% of that number. The term "about" a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[00139] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[00140] The above described embodiments are combinable.

[00141] The following claims further set out particular embodiments of the disclosure. [00142]The present disclosure was supported by - PTDC/BIA-M IC/31561/2017 financially supported by FCT (Foundation for Science and Technology of the Portuguese Ministry of Science and Higher Education) through national funds and co-funded by FEDER under the PT2020 Partnership. OVVieira is funded by the FCT Stimulus of Scientific Employment (CEECINST/00102/2018/CP1567/CT0020). BBM, JConniot and JConde acknowledge the European Research Council Starting Grant (ERC-StG-2019-848325). RC is supported by FCT project PTDC/BIA-MIC/31561/2017. This work is also a result of the GenomePT project (POCI-01-0145- FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciencia e a Tecnologia (FCT).

References

1. Smith, L. & Angarone, M. P. Sexually Transmitted Infections. Urol. Clin. North Am. 42, 507-518 (2015).

2. Grant, J. S. et al. Sexually Transmitted Infections in Pregnancy: A Narrative Review of the Global Research Gaps, Challenges, and Opportunities. Sex. Transm. Dis. 47, (2020).

3. Fichorova, R. N., Tucker, L. D. & Anderson, D. J. The Molecular Basis of Nonoxynol-9- Induced Vaginal Inflammation and Its Possible Relevance to Human Immunodeficiency Virus Type 1 Transmission. J. Infect. Dis. 184, 418—428 (2001).

4. Feldblum, P. J. et al. SAVVY Vaginal Gel (C31G) for Prevention of HIV Infection: A Randomized Controlled Trial in Nigeria. PLoS One 3, el474 (2008).

5. Peterson, L. et al. SAVVY® (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Ghana. PLoS One 2, el312 (2007).

6. Krebs, F. C., Miller, S. R., Malamud, D., Howett, M. K. & Wigdahl, B. Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. Antiviral Res. 43, 157-173 (1999).

7. Inacio, A. S. et al. In Vitro Surfactant Structure-Toxicity Relationships: Implications for Surfactant Use in Sexually Transmitted Infection Prophylaxis and Contraception. PLoS One 6, el9850- (2011).

8. S, I. A. et al. In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents. Antimicrob. Agents Chemother. 60, 3323-3332 (2016).

9. Wang, X. et al. Phenotypic and molecular characterization of Streptococcus agalactiae colonized in Chinese pregnant women: predominance of ST19/III and ST17/III. Res. Microbiol. 169, 101-107 (2018).

10. J., C. B. et al. Comparative Safety Evaluation of the Candidate Vaginal Microbicide C31G. Antimicrob. Agents Chemother. 49, 1509-1520 (2005). 11. Fichorova, R. N. et al. Interleukin (IL)-l, IL-6, and IL-8 Predict Mucosal Toxicity of Vaginal Microbicidal Contraceptives!. Biol. Reprod. 71, 761-769 (2004).

12. Passmore, J. -A. S., Jaspan, H. B. & Masson, L. Genital inflammation, immune activation and risk of sexual HIV acquisition. Curr. Opin. HIV AIDS 11, (2016).

13. Pinto, M. et al. Fifteen years of a nationwide culture collection of Neisseria gonorrhoeae antimicrobial resistance in Portugal. Ear. J. Clin. Microbiol. Infect. Dis. 39, 1761-1770 (2020).

14. EUCAST reading guide for broth microdilution, v. 3.0, Editor. (2021).

15. Cartwright, C. P., Stock, F. & Gill, V. J. Improved enrichment broth for cultivation of fastidious organisms. J. Clin. Microbiol. 32, 1825-1826 (1994).

16. Llarena, A.-K. et al. INNUENDO: A cross-sectoral platform for the integration of genomics in the surveillance of food-borne pathogens. EFSA Support. Publ. 15, 1498E (2018).

17. Aziz, R. K. et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics 9, 75 (2008).

18. Darling, A. E., Mau, B. & Perna, N. T. progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement. PLoS One 5, elll47- (2010).

19. Arndt, D. et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res. 44, W16-W21 (2016).

20. Johansson, M. H. K. et al. Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder. J. Antimicrob. Chemother. 76, 101-109 (2021).

21. Bortolaia, V. et al. ResFinder 4.0 for predictions of phenotypes from genotypes. J. Antimicrob. Chemother. 75, 3491-3500 (2020).

22.Alcock, B. P. et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 48, D517-D525 (2020).

23. Hunt, M. et al. ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads. Microb. genomics 3, e000131-e000131 (2017).

24. W., D. et al. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistances Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains. J. Clin. Microbiol. 55, 1454-1468 (2017).

25. Wood, D. E., Lu, J. & Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 20, 257 (2019).

26. Harris, S. R. et al. Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey. Lancet Infect. Dis. 18, 758-768 (2018).

27. Mei, H., Sobhan, N., Christopher, D. & A., N. R. High-Level Chromosomally Mediated Tetracycline Resistance in Neisseria gonorrhoeae Results from a Point Mutation in the rpsJ Gene Encoding Ribosomal Protein S10 in Combination with the mtrR and penB Resistance Determinants. Antimicrob. Agents Chemother. 49, 4327-4334 (2005).

28. Unemo, M. et al. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization. J. Antimicrob. Chemother. 71, 3096-3108 (2016).

29. Magnus, U. & M., S. W. Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future. Clin. Microbiol. Rev. T1 , 587-613 (2014). 30. Nolte, O. et al. Description of new mutations in the rpoB gene in rifampicin-resistant Neisseria meningitidis selected in vitro in a stepwise manner. J. Med. Microbiol. 52, 1077- 1081 (2003).

31. Calado, J. et al. Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates from men who have sex with men. Int. J. Infect. Dis. 79, 116-122 (2019).

32. Hayes, K., O'Halloran, F. & Cotter, L. A review of antibiotic resistance in Group B Streptococcus: the story so far. Crit. Rev. Microbiol. 46, 253-269 (2020).

33. Da Cunha, V. et al. Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline. Nat. Commun. 5, 4544 (2014).

34. Jansen, G., Barbosa, C. & Schulenburg, H. Experimental evolution as an efficient tool to dissect adaptive paths to antibiotic resistance. Drug Resist. Updat. 16, 96-107 (2013).

35. Harrison, O. B. et al. Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance. J. Infect. 73, 578-587 (2016).

36.0eschger, T. M. & Erickson, D. C. Visible colorimetric growth indicators of Neisseria gonorrhoeae for low-cost diagnostic applications. PLoS One 16, e0252961 (2021).

37. Foerster, S., Unemo, M., Hathaway, L. J., Low, N. & Althaus, C. L. Time-kill curve analysis and pharmacodynamic modelling for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae. BMC Microbiol. 16, 216 (2016).

38. Masaya, T., Yuko, Y., Hideyuki, F., Mitsuru, Y. & Takashi, D. Cultivation of Neisseria gonorrhoeae in Liquid Media and Determination of Its In Vitro Susceptibilities to Quinolones. J. Clin. Microbiol. 43, 4321-4327 (2005).

39. Verhoeven, E. et al. Construction and optimization of a ?NG Morbidostat? - An automated continuous-culture device for studying the pathways towards antibiotic resistance in Neisseria gonorrhoeae [version 2; peer review: 2 approved]. FlOOOResearch 8, (2020).

44. Inacio, A. S. et al. Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis. J. Antimicrob. Chemother. 71, 641-654 (2016).

Claims

C L A I M S

1. Topical pharmaceutical composition for use in the treatment of skin and/or mucosal infections or disorders wherein said topical composition comprises: a pharmaceutically effective amount of a dodecylpyridinium salt and a suitable hydrogel; wherein the topical pharmaceutical composition comprises a viscosity ranging from 1 to 20 Pascal seconds (Pa.s) when measured at a shear rate of 25s ¹ and 37 °C; wherein the dodecylpyridinium salt is dodecylpyridinium bromide; wherein the amount of dodecylpyridinium salt ranges from 400-900 μM.

2. Composition for use according to any of the previous claims wherein the amount of dodecylpyridinium salt is less than 1000 μM.

3. The composition for use according to any of the previous claims wherein the amount of dodecylpyridinium salt ranges from 400-900 μM; preferably 600-800 μM.

4. The composition for use according to any of the previous claims wherein the viscosity of the composition ranges from 1 to 15 Pascal seconds; preferably ranges from 1 to 10 Pascal seconds; when measured at a shear rate of 25s ¹ and 37 °C.

5. The composition for use according to any of the previous claims wherein the suitable hydrogel comprises a biocompatible polymer selected from a list consisting of: cellulose derivatives; polysaccharides; alginates; chitosans; polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid; gellan gum; carbopol; polyethylene glycol; acrylamide; hyaluronic acid; collagen, dextran, fibrin, gelatin, glycogen, heparin, poly(P-aminoester), poly(caprolactone), matrigel, polyhydroxyethylacrylate, poly(hydroxyethyl methacrylate), poly(Nisopropylacrylamide), poly(glycolic acid), poly(lactic acid), poly(lactic acid-glycolicacid), oligo(poly(ethylene glycol)fumarate), poly(vinyl acid)and/or biocompatible organic polymers andcopolymers, or mixtures thereof.

6. The composition for use according to any of the previous claims wherein the polymer is a cellulose-derivative; preferably the cellulose derivative is selected from hydroxyethylcellulose, hydroxypropylmethylcellulose, or copolymers or mixtures thereof; more preferably hydroxypropylmethylcellulose.

7. The composition for use according to any of the previous claims wherein the hydrogel comprises 1-10 % (w/w) of polymer; preferably 1-5 % (w/w); more preferably 1-3 % (w/w).

8. The composition for use according to any of the previous claims, further comprising an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a cell, a buffer, a growth factor, a second suitable hydrogel or combinations thereof.

9. Composition for use according to any of the previous claims for use in the prevention or treatment of skin and/or mucosal infections or disorders caused by a bacterium.

10. Composition for use according to any of the previous claims for use in the prevention or treatment of sexually transmitted infections and/or perinatal infections caused by a bacterium.

11. Composition for use according to any of the previous claims 9-10 wherein the bacterium is selected from the group consisting of: Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycoplasma genitalium, Gardnerella vaginalis, Treponema pallidum, Haemophilus ducreyi, Klebsiella granulomatis (or Calymmatobacterium granulomatis) or mixtures thereof; preferably Neisseria gonorrhoeae.

12. The composition for use according to any of the previous claims, wherein the composition is administered topically to a skin area or to a mucosal area; preferably the mucosal area is selected from the group consisting of vaginal, rectal, and buccal cavities; preferably the mucosal area is on the vulva, perianal, the lining of the anus and on the penis.

13. The composition for use according to any of the previous claims wherein the composition is an intravaginal composition.