Enhanced activity of octiset® versus octenidine against polymicrobial bacterial vaginosis biofilms in vitro and ex vivo

Abstract

Background

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and is strongly associated with polymicrobial biofilms, which contribute to antimicrobial tolerance and high recurrence rates. New strategies targeting BV-associated biofilms are therefore needed. This study evaluated the antimicrobial and antibiofilm activity of octenidine dihydrochloride/phenoxyethanol (Octiset®) compared with octenidine dihydrochloride (octenidine) alone in in vitro and ex vivo BV biofilm models.

Methods

Planktonic cultures and biofilms of BV-associated species (Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia, Peptostreptococcus anaerobius and Mobiluncus curtisii) were established under anaerobic conditions. Planktonic suspensions were exposed for 24 h to octiset® (50%, 25%) or octenidine (0.1%, 0.05%). Single-species and polymicrobial biofilms were formed for 24 h and treated with octiset® or octenidine up to 12 h. Additionally, polymicrobial biofilms were established on reconstructed human vaginal epithelium (SkinEthic™ HVE) and treated for 8 h. Antimicrobial efficacy was assessed by CFU quantification. Recovery experiments were performed to determine whether residual culturable cells persisted after treatment, by incubating samples in fresh medium for up to 72 h and subsequently quantifying CFUs. Antimicrobial activity was also tested against species associated with healthy vaginal microbiota, by assessing vaginal lactobacilli susceptibility to both compounds.

Results

Both compounds showed time- and concentration-dependent activity. Octiset® consistently demonstrated superior killing compared to octenidine, including eradication of several single-species biofilms. Polymicrobial biofilms showed increased tolerance; however, extended exposure (≥8 h) reduced CFUs below detection for both agents, with no regrowth observed after 72 h. In the ex vivo model, both treatments reduced biofilm growth below the limit of detection. All tested lactobacilli also showed susceptibility to both compounds.

Conclusion

Octiset® and octenidine exhibited strong antibiofilm activity against BV-associated polymicrobial biofilms in vitro and ex vivo, supporting their potential as biofilm-targeted BV treatment approaches.

1. Introduction

Bacterial vaginosis (BV) is the most prevalent vaginal infection in women of reproductive age [1,2] and remains a major clinical challenge due to its high recurrence rates [3]. From the microbiological standpoint, BV is characterized by a shift from a Lactobacillus-dominated vaginal microbiota to a polymicrobial community enriched in several anaerobic bacteria [4], including, among many other species, Gardnerella spp. [5,6], Fannyhessea vaginae [7,8], Mobiluncus spp. (Li et al., 2022), Prevotella bivia [8], and Peptostreptococcus anaerobius [9]. Beyond symptomatic discomfort, BV is associated with adverse reproductive outcomes [10] and increased susceptibility to sexually transmitted infections [11,12], highlighting its broader clinical relevance.

A key feature underlying BV persistence is the formation of structured polymicrobial biofilms on the vaginal epithelium, where Gardnerella species typically predominate [13]. These biofilms provide a protective environment that enhances bacterial survival [3,14], promotes synergistic interactions among BV-associated species [15], and significantly increase tolerance to antimicrobial agents [16]. As a result, standard antibiotic therapies such as metronidazole or clindamycin frequently fail to fully eradicate biofilm-associated bacteria, contributing to relapse and chronic colonization [17,18]. In parallel, growing concerns regarding antimicrobial resistance and the impact of antibiotics on the beneficial vaginal microbiota [19] further reinforce the need for alternative therapeutic approaches.

Antiseptic compounds with broad-spectrum antimicrobial activity, such as octenidine, represent a potential strategy for BV management, particularly if they demonstrate efficacy against established biofilms [20]. Octenidine dihydrochloride is a well-known cationic antiseptic with established clinical use in dermatological antisepsis [21], wound care [22], and the treatment of mucous membrane [23]. Its advantageous safety profile stems from the fact that the active ingredient is not absorbed through the skin [[24], [25], [26], [27]]. Furthermore, octenidine provides a persistent antimicrobial effect lasting at least 24 to 48 h [28,29], acting rapidly through non-specific physical interactions with the lipid membranes of a broad-spectrum pathogens, including Gram-positive [30] and Gram-negative bacteria [31]. As far as we are aware, development of resistance to octenidine dihydrochloride has not yet been observed, and it remains highly effective even against drug-resistant strains. Due to the physical mechanism of action, the development of resistance is unlikely. While commercial formulation containing phenoxyethanol (octiset®) may enhance antimicrobial performance through synergistic effects [32], systematic evaluations against BV-relevant polymicrobial biofilms in physiologically relevant models remain limited.

In this study, we investigated the antimicrobial and antibiofilm activity of octiset® and octenidine alone against key BV-associated bacteria in planktonic and biofilm states, using both in vitro multispecies biofilm systems and an ex vivo reconstructed human vaginal epithelium model. This approach aims to provide relevant evidence for the potential of octenidine-based formulations as biofilm-targeted interventions for BV.

2. Materials and methods

2.1. Bacterial strains

Bacterial vaginosis-associated bacteria (BVAB) used in this study were obtained from the American Type Culture Collection (ATCC) and the Culture Collection University of Gothenburg (CCUG), namely Gardnerella vaginalis ATCC 14018, Fannyhessea vaginae ATCC BAA-55, Mobiluncus curtisii ATCC 35241, Peptostreptococcus anaerobius ATCC 27337, and Prevotella bivia CCUG 34046. In addition, representative protective vaginal Lactobacillus species were included, namely Lactobacillus crispatus EX533959VC06, Lactobacillus jensenii CCUG 44492, and Lactobacillus gasseri CCUG 44075. The cultures were preserved in Brain Heart Infusion medium (Liofilchem, Italy) supplemented with 23% glycerol (Panreac, Darmstadt, Germany) at −80 °C.

2.2. Culture conditions and inoculum preparation

Bacteria strains were inoculated on Columbia Blood Agar (CBA, Oxoid, Basingstoke, UK) supplemented with 5% (v/v) defibrinated horse blood (Thermo Fisher Scientific, Lenexa, KS). Plates were incubated at 37 °C for 48 h under anaerobic conditions generated by AnaeroGen™ sachets (Thermo Fisher Scientific, Gloucester, UK). Before each assay, a pre-inoculum of each strain was prepared in New York City III (NYCIII) medium supplemented with horse serum heat inactivated, as previously described [33]. Each strain inoculum was incubated for 24 h at 37 °C under anaerobic conditions.

2.3. Antiseptic agents’ preparation

Octiset®, also known as octenisept®, an approved drug (0.1% octenidine dihydrochloride and 2% phenoxyethanol; Schülke & Mayr GmbH, Norderstedt, Germany) was freshly prepared before each experiment, by diluting in NYCIII medium to the desired concentrations. Octenidine dihydrochloride (98%; Thermo Fisher Scientific, Gloucester, UK) was first diluted in sterile ultrapure water (pH 6.5) to obtain a stock concentration of 0.4% that was kept at 4 °C. Before each experiment, this solution was further diluted in NYCIII medium to the required working concentrations.

2.4. Susceptibility of planktonic cells

BVAB and protective vaginal lactobacilli viability following antiseptic exposure was evaluated, as described below. The optical density (OD) of each inoculum was measured at 620 nm and adjusted to a final concentration of 1 × 10⁷ CFU/mL, as previously optimized [34,35]. A volume of 500 μL of each bacterial suspension was added to 24-well plate (NEST Biotechnology, Wuxy, China) containing octiset® (50% and 25%) and octenidine (0.1% and 0.05%). The plates were incubated under anaerobic conditions at 37 °C for 6 h or 24 h. At each time point, the content of each well was resuspended and the culturable cells were quantified at 0 h, 6 h and 24 h, using the colony-forming unit (CFU) enumeration method, as described below. All assays were performed at least three independent times with duplicates.

2.5. Biofilm formation and antiseptic treatment

2.5.1. In vitro single- and mixed-species biofilms

BVAB suspensions were adjusted to 1 × 10⁷ CFU/mL in NYCIII broth by measuring OD. Biofilms were seeded and developed in 24-well culture plates. For single-species biofilms, 1 mL of a 1 × 10⁷ CFU/mL suspension of each strain was inoculated in each well. For mixed-species biofilms, the five species were combined in a 1:1 ratio for a final concentration of 1 × 10⁷ CFU/mL. Plates were incubated at 37 °C for 24 h under anaerobic conditions. After this time, the biofilms were washed once with 0.9% NaCl and 1 mL of octiset® (50% and 25%) and octenidine (0.1% and 0.05%) was added to the biofilms. Biofilms were subjected to treatment with the indicated antiseptic solutions under multiple exposure times and concentration conditions, as defined for each experimental setup. Detailed treatment parameters are provided in the corresponding figure legends and Supplementary Material. After treatment, the biofilms cells were quantified using the CFU method, as described below. All assays were performed at least three independent times with duplicates.

2.5.2. Ex vivo polymicrobial biofilms on reconstructed human vaginal epithelium (HVE)

These experiments used SkinEthic™ HVE (reconstructed model of a human vaginal epithelium), obtained from L’Oréal (EpiSkin S.A.S., Lyon, France). Each species inoculum concentration was adjusted to 9 × 10⁷ CFU/mL by measuring OD. The tissues were placed on a 24-well plate and the bacterial suspensions were added to the tissues for a final volume of 500 μL and final concentration of 1 × 10⁷ CFU/mL. The plate was incubated at 37 °C under anaerobic conditions for 16 h to form the biofilm. Following the 16 h incubation, the culture medium was gently removed from the tissues and biofilms were washed once with sterile 0.9% NaCl to remove non-adherent cells. The antiseptic compounds were then added to the biofilms, only the highest effective concentrations were selected: 50% of octiset® and 0.1% of octenidine dihydrochloride. The tissues with agents were incubated with the compounds for 8 h at 37 °C under anaerobic conditions. The tissues from the 24 h-biofilm control were used for quantification by CFUs and qPCR. The medium from the tissues was removed, the tissues were washed once with 0.9% NaCl, and 500 μL of NYCIII was added. The cells were detached from the tissues using 10 s of sonication at 33% amplitude. The content of the well was thoroughly resuspended and quantified using CFU method. The remaining volume was used for qPCR quantification, as described below. After 8 h treatment, the tissues with agent were treated in the same way as the controls for CFUs quantification. Eight independent experiments were performed with duplicates, and each replicate was quantified by CFUs counts using technical duplicates.

2.6. Culturable cells quantification

BVAB and Lactobacilli growth was evaluated by CFU quantification. From each condition, 50 μL were transferred into microtubes containing medium to obtain a 1:10 dilution; this procedure was repeated sequentially to generate six serial dilutions. From each dilution 10 μL were plated onto CBA plates and the plates were incubated for at least 48 h at 37 °C under anaerobic conditions.

2.7. Post-treatment recovery assay

The recovery assay was performed for single- and mixed-species biofilms, exclusively when no colonies were detected by CFU quantification. The more concentrated dilution from the CFU serial dilutions was incubated at 37 °C for up to 72 h under anaerobic conditions. Every 24 h, 10 μL were plated onto CBA in duplicate, and incubated for up to 72 h at 37 °C under anaerobic conditions. Growth on the plates was assessed every 24 h. The assay was considered complete when growth was detected on both plated duplicates, or when no growth was observed after 72 h of incubation.

2.8. Genomic DNA extraction and quantification by quantitative PCR (qPCR)

Genomic DNA quantification by qPCR was performed as previously described [36], using the DNeasy Ultraclean Microbial Kit (QIAGEN, Hilden, Germany). Briefly, bacterial samples from single- and mixed species biofilms, were centrifuged at 16.000 g for 10 min at 4 °C. The supernatant was removed, and the cell pellet was frozen overnight at −20 °C for preservation. Pellet was resuspended in Power Bead Solution and transferred to a PowerBead Tube with addition of SL solution. Bacterial lysis was performed in a BeadBug 6 Microtube Homogenizer using 2 x 3 cycles of 30 s at 4350 rpm, with samples maintained on ice for 1 min between cycles. The lysate was centrifuged at 10000 g for 30 s. As an exogenous control, 10 μL of 1 ng/μL E. coli gDNA was added before passing the sample through the column. The sample was passed through the column and eluted in 50 μL of DNase free-water afterwards. The final DNA concentration was measured using a Nanodrop spectrophotometer. Before qPCR was performed, the samples were diluted 1:40. qPCR assays were performed on a CFX96™ thermal cycler (Bio-Rad, CA, USA). The total reaction mixture volume was 10 μL, consisting of 5 μL of Xpert Fast SYBR MasterMix (Grisp, Porto, Portugal), 0.5 μL of forward primer, 0.5 μL of reverse primer, 2 μL of DNase free-water, and 2 μL of 1:40 gDNA sample. The thermal cycling parameters were: 95 °C for 3 min followed by 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. A non-template control was included to discard primers contamination. Technical triplicates were performed for all qPCR runs. To assess the efficiency of gDNA extraction and calibrate fluorescence intensity among qPCR runs, a control was used by adding 2 μL of 1:200 of the exogenous control to all independent qPCR runs [37]. Samples were tested for the presence of G. vaginalis, F. vaginae, P. bivia, M. curtisii, and P. anaerobius, using primers previously described [36].

2.9. Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). For planktonic assays, data were analysed using Two-way ANOVA with Dunnett's multiple comparisons test to compare each treatment with the corresponding untreated control. For biofilm assays, Kruskal–Wallis test was applied, followed by Dunn's multiple comparisons test. Differences were considered statistically significant when p < 0.05.

3. Results

3.1. Antimicrobial activity of planktonic assays

To assess the antimicrobial potential of octenidine dihydrochloride and octiset®, we initially attempted to determine the minimum inhibitory concentration using a standard broth microdilution assay. However, during the assay, precipitation of the compounds, possibly due to interactions with the growth media, was observed in the wells, leading to visible deposition. This interfered with the spectrophotometric readings after incubation, generating unreliable optical density measurements and preventing accurate quantification of bacterial growth. Therefore, antimicrobial activity was subsequently evaluated by CFU enumeration in planktonic cultures, by determining the number of CFUs after 6 and 24 h of incubation.

As shown in Fig. 1, both compounds demonstrated antimicrobial activity within 6 h of incubation against all tested bacterial species, including the BVAB (G. vaginalis, F. vaginae, P. bivia, P. anaerobius, and M. curtisii) and the protective vaginal lactobacilli (L. crispatus, L. jensenii, and L. gasseri), with bacterial counts falling below the limit of detection (LOD) across all tested concentrations.

Fig. 1.

Planktonic cultures of BVAB and vaginal lactobacilli were exposed to octiset® (OCT, 50%, 25%) and octenidine (OH, 0.1%, 0.05%), and the results of culturability are represented as Log₁₀ CFU/mL, with error bars representing the standard deviation. All treated conditions were below the LOD (Log₁₀ = 3). Statistical significance relative to the control was determined by two-way ANOVA with Dunnett's multiple comparisons test (p < 0.05).

3.2. Single-species antibiofilm activity

To characterize species-specific susceptibility and optimize conditions for subsequent polymicrobial assays, the antibiofilm activity of octenidine-based antiseptics was first evaluated in single-species biofilms. Similar to the interference observed during MIC determination, crystal violet staining could not be reliably used to assess biofilm biomass, as the tested compounds interfered with the staining/quantification procedure (Supplementary Figure 1). Therefore, antibiofilm activity was also evaluated by CFU enumeration.

Preliminary experiments were conducted using G. vaginalis to optimize exposure times and concentrations. In these assays, octiset® was evaluated at concentrations ranging from 50% to 3.125%, and octenidine dihydrochloride from 0.1% to 0.0125%, with exposure times spanning from 10 min to 24 h. The results demonstrated that although lower concentrations (3.125% octiset® and 0.0125% octenidine) were effective after 24 h of exposure, they were insufficient at shorter time points (10 min and 1 h). In contrast, pronounced antibiofilm activity at early exposure intervals was achieved only with higher concentrations (Supplementary Figure 2). Based on these optimization experiments, subsequent assays were performed using standardized exposure times and concentration ranges that demonstrated efficacy and reflected commercially relevant conditions.

As shown in Fig. 2, both octiset® and octenidine demonstrated a strong antimicrobial activity, which resulted in a reduction in culturable cell counts across all tested species, in comparison to the untreated control. However, this reduction varied significantly between the different species. Notably, P. anaerobius was the most susceptible species, with a very few culturable cells detected after only 1 h of treatment with either antiseptic agents. High susceptibility was also observed in G. vaginalis and F. vaginae, which exhibited pronounced decreases in viability within the same period. Conversely, P. bivia and M. curtisii displayed a more limited response, with M. curtisii identified as the most resilient species. Interestingly, after 4 h of exposure, all tested conditions reduced bacterial counts below the LOD. Thus, 4 h was established as the minimum exposure time required to achieve complete eradication and was used as the reference timepoint for subsequent polymicrobial biofilm experiments.

Fig. 2.

Effect of antiseptic treatments on single-species biofilms. Biofilms of G. vaginalis, F. vaginae, M. curtisii, P. bivia, and P. anaerobius were grown for 24 h and then exposed to octiset® (OCT) and octenidine (OH) for 10 min, 1 h, or 4 h. Bacterial culturability was determined as Log₁₀ CFU/mL, with error bars representing the standard deviation. The LOD was Log₁₀ = 3. Statistical analysis was performed using the two-way ANOVA followed by Dunnett's multiple comparisons test. Statistically significant differences compared to the 24h control group (p < 0.05).

3.3. In vitro polymicrobial activity

Since BV is driven by polymicrobial biofilms, and multispecies communities are known to exhibit increased tolerance to antimicrobial agents compared to single-species biofilms [16], the activity of octenidine dihydrochloride and octiset® was next assessed in a five-species in vitro BV biofilm model. Biofilms were established for 24 h under anaerobic conditions and their baseline composition was characterized by qPCR. As shown in Fig. 3A, the community was dominated by G. vaginalis and F. vaginae, which together accounted for approximately 85–90% of the total bacterial load, while M. curtisii, P. bivia, and P. anaerobius were present at lower proportions. As shown in Fig. 3B, the exposure to both antiseptic solutions for 4 h was not sufficient to fully eradicate the bacterial populations. This highlights that polymicrobial biofilms possess enhanced tolerance relative to their single-species biofilms, as we shown before [36]. Interestingly, after 8 h of exposure, no culturable cells were recovered for any treatment condition, indicating that bacterial counts were reduced below the LOD.

Fig. 3.

In vitro polymicrobial activity of antiseptic compounds on mixed-species biofilms. (A) Biofilm culturability was quantified by CFUs and results are expressed as Log₁₀ CFU/mL, with error bars representing the standard deviation. The LOD was Log₁₀ = 3. (B) Percentage distribution by qPCR of each bacterial species in 24h polymicrobial biofilm. Statistical analysis was performed separately for each treatment time using the Friedman test followed by Dunn's multiple comparisons test, comparing each treatment with the 24 h biofilm control (p < 0.05).

3.4. Ex vivo polymicrobial activity

To further evaluate antiseptic efficacy under more physiologically relevant conditions, the polymicrobial BV biofilm model was established on reconstructed human vaginal epithelium (SkinEthic™ HVE). For these experiments, only the highest concentrations of octenidine dihydrochloride and octiset® were tested. This selection was made in order to better mimic actual application concentrations in patients in clinical practice. Importantly, due to tissue viability constraints [38], biofilms were allowed to develop for 16 h prior to antiseptic challenge, followed by an 8 h exposure period. Under these conditions, the polymicrobial biofilm formed a distinct community composition, characterized by a more balanced species distribution and enrichment of M. curtisii and F. vaginae (Fig. 4A). Furthermore, it is important to acknowledge that the introduction of tissue significantly alters growth conditions compared to in vitro models. Interestingly, in our experimental set up, 8 h of exposure was also sufficient to reduce bacterial counts to levels below the LOD (Fig. 4B).

Fig. 4.

Effect of antiseptic treatment on ex vivo mixed-species biofilms. (A) Composition of 16h polymicrobial biofilm community on reconstructed human vaginal epithelium (SkinEthic™ HVE) as determined by qPCR. (B) Culturability of the polymicrobial biofilm after 8 h of treatment with octiset® (50%) and Octenidine (0.1%), represented by Log₁₀ CFU/mL. The LOD was Log₁₀ = 3. Error bars represent the standard deviation. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test, comparing each treatment with the untreated control (p < 0.05).

3.5. Assessment of bacterial regrowth after treatment

To determine whether bacterial cells persisted after antiseptic exposure, recovery assays were performed following all biofilm treatments described above. Treated biofilms were incubated in fresh antimicrobial-free medium for up to 72 h to evaluate regrowth potential. Under all tested conditions, no culturable cells were recovered, even after prolonged incubation, indicating the absence of detectable regrowth following treatment (Table 1).

Table 1.

Evaluation of bacterial recovery following the recovery assays after 12 h and 24 h of treatment with different antiseptic treatments.

	12h of treatment			24h of treatment
Conditions	24h	48h	72h	24h	48h	72h
Octiset® 50%	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Octiset® 25%	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Octenidine 0.1%	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Octenidine 0.05%	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.

n.d. not detected (bellow the limit of detection).

4. Discussion

The low efficacy of current antibiotics available to treat BV and the high recurrence rates associated with them [39] have led to a search for new therapeutic strategies that target biofilms directly, combining effectiveness with safety [3,40]. Consequently, reducing the bacterial load within the protective biofilm environment is increasingly recognized as a key factor in achieving therapeutic success and mitigating the risk of post-treatment relapse. While previous studies have demonstrated that octiset® and its active component, octenidine dihydrochloride, display a wide spectrum of antimicrobial activity against pathogens such as Acinetobacter [41], Candida auris [42] Enterococcus faecalis [43], Pseudomonas aeruginosa [44], and Candida albicans [45], their efficacy in the specific context of structured, vaginal polymicrobial BV biofilms remains poorly understood.

Our experiments focused on quantifying CFUs under controlled in vitro and ex vivo conditions. Across all tested biofilm models, including single-species and five-species polymicrobial biofilms, exposure to octiset® or octenidine reduced culturable cells below the LOD. As expected for broad-spectrum antiseptic agents, both octiset® and octenidine were also active against representative protective vaginal Lactobacillus species. These results are in line with previously published results for non-vaginal Lactobacillus, namely Lactobacillus acidophilus, Lactobacillus rhamnosus [46], and Lactococcus lactis [47]. Therefore, the antimicrobial activity observed in this study was not selective for BV-associated organisms. While this finding may raise concerns regarding transient disruption of beneficial vaginal microbiota, it should be noted that currently used BV treatments may also affect Lactobacillus populations. Further studies are required to determine whether the potent eradication of BV-associated biofilms observed here ultimately facilitates or hinders re-establishment of a Lactobacillus-dominated vaginal microbiota. In clinical practice, restoration of a Lactobacillus-dominant microbiome is generally considered an important step in reducing recurrence following successful antimicrobial treatment of bacterial vaginosis [48]. Importantly, recovery assays performed for up to 72 h in fresh medium failed to reveal any regrowth, indicating that, under these experimental conditions, the bactericidal effect of these agents is highly potent. However, since the recovery period was defined as 3 days, our findings do not allow us to draw conclusions about total eradication or possibility of regrowth beyond this period. In a pilot clinical study, Swidsinski and colleagues used FISH-based detection to investigate Gardnerella cells following antiseptic treatment and reported that no detectable biofilm was present immediately after treatment (Swidsinski et al., 2015); this is consistent with our experimental observations. However, in a subgroup of patients, biofilm structures were detected again during follow-up examinations within 6 months. It remains unclear whether these findings reflected biofilm persistence, regrowth from residual cells, or reinfection. These results may indicate the potential for renewed biofilm formation or persistent bacterial reservoirs. FISH detects all cells irrespective of culturability, including dormant or non-dividing cells [49], whereas our CFU-based approach reflects only the cultivable fraction of the population [50]. As a result, while Swidsinski's work indicates persistence of biofilm structure and potentially non-culturable cells in clinical BV, our results show complete elimination of cultivable cells under sustained exposure in controlled models. It is important to emphasize that methodological differences limit direct comparison between the two studies. Nevertheless, our in vitro and ex vivo findings highlight the strong efficacy of sustained antiseptic exposure under controlled conditions, providing a valuable experimental foundation for understanding treatment effects, while long-term clinical recurrence dynamics require further investigation.

Although previous studies show good tolerability to octiset® [23,51], we also attempted to determine cytotoxicity under of tested conditions. However, we noticed that both compounds reacted with the colorimetric MTS-based viability assay, generating colorimetric signals independent of cellular metabolism (Supplementary Table 1). As a result, epithelial viability could not be reliably quantified under the conditions tested. Another limitation of this work was related to the use of strictly anaerobic conditions required for the growth of BV-associated obligate anaerobic bacteria. While these conditions were essential to support the establishment and maintenance of polymicrobial biofilms, they may not be optimal for sustaining long-term viability of the reconstructed vaginal epithelium. Since epithelial viability was not directly assessed in this setting, it remains unclear to what extent the tissue remained metabolically active throughout the experimental period. However, morphological examination of the tissue following anaerobic incubation has previously suggested preservation of epithelial architecture under similar experimental conditions [34]. Consequently, the ex vivo model should be interpreted primarily as a biologically relevant surface for biofilm establishment and antimicrobial testing rather than a system for investigating host-pathogen interactions, and therefore does not fully capture the complexity of the vaginal microenvironment, including host immune responses, hormonal influences, mucus dynamics, and interactions with the resident microbiota.

To further characterize the biofilm communities in our models, qPCR analyses were used to assess species composition. In vitro biofilms were dominated by G. vaginalis, whereas ex vivo biofilms on human vaginal epithelium exhibited a more balanced microbial community, with increased contributions from F. vaginae and M. curtisii. These differences likely reflect the influence of the tissue environment on species-specific growth and adhesion [50]. Importantly, despite these compositional variations, exposure to octiset® and octenidine effectively reduced cultivable cells below the LOD in both models. Thus, the potent antibiofilm activity of these agents appears largely independent of biofilm composition, reinforcing their broad efficacy across polymicrobial biofilms.

Importantly, our study provides mechanistic insight into the relative contributions of the formulation and its active component. By comparing octiset® and octenidine dihydrochloride directly, we confirmed that the potent bactericidal effect is driven primarily by octenidine. Despite the potent antimicrobial results obtained under our tested conditions, it is relevant to note that an increase in tolerance was observed when polymicrobial biofilms were tested, in comparison with single-species biofilms, providing further evidence of synergistic effects between these bacteria might be the crucial reason why so many therapeutic options fail, on the long term [9]. Interestingly, F. vaginae, known to be resistant to many antibiotics [16,52], also showed higher tolerance to octenidine dihydrochloride, at the lower concentrations tested (0.05%), but this did not compromise the complete reduction of viable counts in the polymicrobial assays performed at the higher concentration, which are even lower than the concentrations applied with patients.

Overall, this study demonstrates that octenidine dihydrochloride and octiset® exhibit strong antimicrobial activity against key bacterial species associated with BV, including within polymicrobial biofilm structures. Both agents significantly reduced culturable cell counts in planktonic cultures, and although microbial tolerance increased in biofilms, exposure at higher concentrations resulted in reductions of bacteria culturability below the LOD. These findings highlight the potent antibiofilm potential of octenidine-based formulations and support further investigations to optimize dosing, exposure times, and application strategies in models that closely mimic the vaginal environment.

CRediT authorship contribution statement

Filipa O. Castro: Investigation, Writing – original draft. Taynara Mulinde: Investigation, Writing – review & editing. Lúcia G.V. Sousa: Investigation, Writing – review & editing. Pascal Bartling: Conceptualization, Writing – review & editing. Gwen Zwingenberger: Conceptualization, Writing – review & editing. Nuno Cerca: Conceptualization, Funding acquisition, Methodology, Writing – original draft.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nuno Cerca reports financial support was provided by Schülke & Mayr GmbH. Nuno Cerca reports financial support was provided by Portuguese Foundation for Science and Technology (FCT). Pascal Bartling reports a relationship with Schülke & Mayr GmbH that includes: employment. Gwen Zwingenberger reports a relationship with Schülke & Mayr GmbH that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.