The following assays utilized T. vaginalis ATCC 30236 and a fresh clinical isolate, TV-LACM6, obtained from the Laboratório de Análises Clínicas e Toxicológicas, Faculdade de Farmácia UFRGS, Brazil (approved by the UFRGS Research Ethical Committee under authorization number 18923). These isolates were selected based on their distinct characteristics; TV-LACM6 exhibits higher hemolytic activity and cytolytic capacity than ATCC 30236 (Menezes et al., 2017). Trophozoites were cultured in Trypticase Yeast Maltose (TYM) medium (Diamond, 1957), supplemented with 10% (v/v) adult bovine serum (ABS) and penicillin/streptomycin, for 24 h under microaerophilic conditions at 37°C. During logarithmic growth, the T. vaginalis suspension was centrifuged and resuspended in Man-Rogosa-Sharpe broth (MRS) - chosen based on the results explained in the following sections - (Sigma-Aldrich) (De Man et al., 1960) supplemented with 10% ABS. Trophozoite densities of 1 x 10⁶ (TV1) and 1 x 10⁵ (TV2) trophozoites/mL were tested and adjusted using a Neubauer hemocytometer with Trypan-blue exclusion dye (0.4%) under an optical microscope (Nikon TE 2000-U Eclipse). MRS medium is composed of peptone, meat extract, yeast extract, and glucose, which provide essential nutrients through enzymatic digestion. Growth stimulation is induced by polyoxyethylenesorbitan monooleate, magnesium sulfate, and manganous sulfate. Additionally, ammonium citrate helps maintain a low pH, promoting the growth of Lactobacillus while inhibiting the proliferation of other microorganisms.

C. albicans (ATCC 24433) was cultured on Sabouraud dextrose agar for 24 h at 37°C, while L. crispatus (CCT 7595), obtained from the Tropical Culture Collection of André Tosello Foundation (PR, Brazil), was grown on MRS agar under the same conditions. The suspensions of C. albicans and L. crispatus were adjusted to optical densities (OD) of 0.110 and 0.160, respectively, at 620 nm using a Multiskan™ FC Microplate Photometer (Thermo Fischer Scientific, USA). Each suspension was then serially diluted 1:10 in saline, resulting in five different density ranges. The maximum densities used in the co-culture assay were 3.33 x 10⁶ CFU/mL for C. albicans (CA1) and 5.53 x 10⁷ CFU/mL for L. crispatus (LC1), with minimum densities of 3.33 x 10² CFU/mL (CA5) and 5.53 x 10³ CFU/mL (LC5), respectively.

The media tested for their capacity to support the growth of all three microorganisms in monoculture over 24 h of incubation included TYM, Sabouraud dextrose broth (SDB), and MRS, all supplemented with only 10% ABS, or MRS supplemented with 10% ABS and 20 µM of fluconazole, and RPMI-1640 (Sigma-Aldrich) medium supplemented with 20% fetal bovine serum (FBS). The evaluation of growth was based on the presence or absence of microorganisms, as observed in microscopy slide cultures using Trypan blue dye exclusion.

To determine the optimal density of T. vaginalis ATCC 30236, C. albicans ATCC 24433, and L. crispatus CCT 7595 for co-culture, a checkerboard pattern was created on 96-well culture plates. In this experimental design, one species was tested at a fixed density while the other species was tested across different density ranges. Specifically, T. vaginalis was tested at two different levels (1.00 x 10⁶ and 1.00 x 10⁵ trophozoites/mL, TV1 and TV2), while C. albicans (3.33 x 10⁶ to 3.33 x 10² CFU/mL, CA1 to CA5) and L. crispatus (5.53 x 10⁷ to 5.53 x 10³ CFU/mL, LC1 to LC5) were tested across five different density ranges. This approach allowed for the evaluation of various combinations to determine the optimal co-culture density. Monocultures, dual co-cultures, and triple co-culture of T. vaginalis isolates, C. albicans, and L. crispatus were employed, with all combinations detailed in Supplementary Table 2 . Each adjusted microbial suspension was added in a volume of 50 μL to each well, resulting in a final volume of 200 μL. The plates were then incubated at 37°C for 24 h. All combinations used are shown in Supplementary Table S2 .

For standardizing the co-culture of TV-LACM6 (at 1.00 x 10⁶ trophozoites/mL), C. albicans was maintained at 3.33 x 10⁴ CFU/mL, while L. crispatus varied at the first two densities 5.53 x 10⁷ (LC1, OD 0.160) and 5.53 x 10⁶ CFU/mL (LC2, dilution 1:10).

After 24 h of co-culture incubation, the contents of each well were diluted 1:1000 in saline. For quantification of C. albicans colony formation units (CFU/mL), the diluted samples were inoculated onto Sabouraud agar plates. To assess L. crispatus CFU/mL, the samples were inoculated onto MRS agar supplemented with 20 µM fluconazole, a concentration that inhibits C. albicans growth. Both sets of agar plates were incubated at 37°C for 24 h. The viability of T. vaginalis was evaluated using a Neubauer Hemocytometer and Trypan-blue exclusion dye (0.4%) under an optical microscope (Nikon TE 2000-U Eclipse).

For subsequent assays, T. vaginalis isolates were adjusted to a density of 1.00x10⁶ trophozoites/mL (TV1) and C. albicans was adjusted to an initial OD of 0.110 before being diluted 1:100 in saline (CA3). L. crispatus was adjusted to an initial OD of 0.160 (LC1) and then diluted 1:10 in saline (LC2). These two suspensions of L. crispatus were used in co-cultures of T. vaginalis isolates TV-LACM6 and ATCC30236, respectively.

After standardizing the optimal density for each microorganism, the growth kinetics were assessed. T. vaginalis isolates, C. albicans, and L. crispatus strains were adjusted as described in Section 2.5. To determine the growth curve, each adjusted suspension was added in a volume of 125 μL to each microtube, resulting in a final volume of 500 μL. The growth curve was evaluated in monocultures and co-cultures of the three microorganisms. OD readings were taken at 2, 4, 6, 8, 24, 48, and 72 h. Microbial viability was assessed at these same time points using a hemocytometer for trophozoite viability and OD readings for C. albicans and L. crispatus.

MRS broth supplemented with 10% ABS was prepared with an initial pH of 6.0. T. vaginalis isolates, C. albicans, and L. crispatus strains were adjusted according to the procedure outlined in Section 2.5. To measure the pH of the medium in co-cultures, pH strips were utilized. Each adjusted microbial suspension was added in a volume of 125 μL to each microtube, resulting in a final volume of 500 μL. pH measurements were taken after 24h of incubation, including monocultures, co-cultures of T. vaginalis isolates or C. albicans strain with two densities of L. crispatus, and triple co-cultures.

T. vaginalis isolates, C. albicans, and L. crispatus strains were prepared as described in Section 2.5. The effect of metronidazole was analyzed at concentrations ranging from 100 μM to 0.78 μM on monocultures of T. vaginalis isolates ATCC 30236 and TV-LACM6, as well as on the co-culture of these isolates with L. crispatus at 5.5x10⁶ (dilution 1:10) and 5.5 x 10⁷ CFU/mL (OD 0.160), respectively, and with C. albicans. The effect of fluconazole was examined at concentrations from 100 μM to 0.78 μM on the monoculture of C. albicans and on the co-culture of C. albicans with T. vaginalis isolate ATCC 30236 and L. crispatus at 5.5 x 10⁶ CFU/mL. The effect of penicillin/streptomycin was analyzed at concentrations ranging from 100 μM to 0.78 μM on the monoculture of L. crispatus at 5.5 x 10⁷CFU/mL and on the co-culture of L. crispatus with T. vaginalis isolate TV-LACM6 and C. albicans. After 24 h of incubation at 37 °C, microbial viability was assessed using the methods described in Section 2.5.

To create an in vitro environment that closely resembles the in vivo conditions, a monolayer of vaginal cells from the HMVII lineage (human vaginal epithelial melanoma) was co-incubated with a co-culture of T. vaginalis isolates, C. albicans, and L. crispatus. HMVII cells at density of 2.0 x10⁵ cells/mL were seeded in 96-well culture plates and incubated at 37°C with 5% CO₂ until they formed a confluent monolayer. The cells were cultured in RPMI medium supplemented with 20% FBS. Subsequently, T. vaginalis isolates, C. albicans, and L. crispatus strains were prepared as described in Section 2.5, with the modification that the medium utilized was RPMI supplemented with 20% FBS. These adjusted microbial suspensions were then added to each well containing the HMVII monolayer. To study the interactions of microorganisms with vaginal epithelial cells, we examined all monoculture and co-culture conditions of T. vaginalis, C. albicans, and L. crispatus at the standard densities described in section 2.5 (TV1, CA3 and LC1 or LC2).

After the co-incubation of HMVII cell with a co-culture of T. vaginalis isolates, C. albicans, and L. crispatus as described in Section 2.9, an assay was performed to detect the lactate dehydrogenase (LDH) enzyme released by epithelial vaginal cells. This assay aimed to evaluate the cytolytic capacity of T. vaginalis isolates in co-culture compared to monoculture. The Cytotox-one Homogeneous Membrane Integrity Assay (Promega, USA) was used for this purpose. The 96-well culture plates were incubated at 37°C with 5% CO₂ for 6 h. Subsequently, LDH release was measured according to the manufacturers’ instructions. The results were calculated as described by Rigo et al. (2023).

Blood samples obtained from volunteer donors (approved by the UFRGS Research Ethical Committee under authorization number 6742238) were centrifuged (2500 x g, 10 min) to separate the plasma, which was then discarded. The erythrocytes were washed three times with phosphate-buffered saline (PBS) at pH 7.0 and adjusted to a density of 5.0 x 10⁷ erythrocytes/mL. To determine hemolytic capacity, each adjusted microbial suspension (as described in Section 2.5) was added in a volume of 125 μL to each microtube, resulting in a final volume of 500 μL. Hemolytic capacity was measured after 24 h at 37°C following the co-incubation of erythrocytes with T. vaginalis isolates (monoculture), a co-culture of T. vaginalis isolates with either C. albicans or L. crispatus (dual co-culture), a co-culture with C. albicans and L. crispatus (dual co-culture), and T. vaginalis isolates with C. albicans and L. crispatus (triple co-culture). Controls, procedures for measuring hemolysis, and result calculations were performed as described by Menezes et al. (2017).

The biofilm biomass of C. albicans and L. crispatus was compared with that of C. albicans and L. crispatus in co-culture. This evaluation was conducted using the crystal violet assay, with 0.4% crystal violet, where absorbance was measured at 570 nm, according to the protocol by Trentin et al. (2011) Briefly, microorganisms were adjusted as described in Section 2.5. The combinations in the 96-well microtiter plate were as follows: only C. albicans, only L. crispatus, C. albicans with T. vaginalis isolates, C. albicans with L. crispatus at two densities, co-culture of all microorganisms, and a sterility control (MRS medium and saline solution). The values obtained for the sterility control were subtracted from all readings, and the results were expressed as a percentage of biofilm formation, with the biofilm formed by only C. albicans and L. crispatus representing 100%.

To evaluate the metabolic viability of biofilm cells formed by the adjusted microorganisms, as described in Section 2.5, the combinations in the 96-well microtiter plate were prepared as outlined in section 2.13. After 24 h, the contents of each well were discarded, and the wells were washed three times with saline, following the method of de Oliveira Dembogurski et al. (2018). Briefly, MTT solution (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) at 0.3 mg/mL was added, and the plates were incubated for 1 h 30 min at 37°C. After incubation, MTT was removed, and the wells were washed before adding DMSO (dimethyl sulfoxide). After 20 min, absorbance was measured at 570 nm. The results were calculated as described in Section 2.13.

The morphological transformation of C. albicans from yeast to hyphae in co-culture was evaluated following the method described by Toenjes et al. (2005), with some modifications. Briefly, microorganisms were adjusted as described in 2.5 section. The combinations in the 96-well microtiter plate were as follows, only C. albicans, C. albicans with T. vaginalis isolates, C. albicans with L. crispatus at two densities, and triple co-cultures. The microplate was incubated for 24h at 37°C. After incubation, all combinations were analyzed by counting the number of yeast cells versus hyphae under a microscope using a Neubauer hemocytometer. The results were expressed as the hyphae formation rate relative to the total number of Candida cells.

Co-cultures of T. vaginalis isolates, C albicans and L. crispatus, as well as monocultures of each microorganism, were prepared according to Section 2.5 section. After 24 h, prior to fixation with 2.5% (v/v) glutaraldehyde, the suspensions were washed three times with 1X PBS over 2 h 30 min. Subsequently, a second set of three washes with PBS was performed before post-fixation with 1% osmium tetroxide for 2 h. The samples were then placed on a circular cover slip coated with 0.1% poli-L-lysine. The cover slips were dehydrated in a graded acetone series (30, 50, 70, 80, 95 and 100° GL). Critical point drying was conducted using CO_2, and the samples were sputter-coated with gold particles. The metalized samples were then analyzed using a Zeiss EVO MA10 scanning electron microscope. The resulting images were analyzed using the ImageJ software (v1.54m).

The data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using the Student’s t-test to compare two groups (monoculture and co-culture) with GraphPad Prism software version 8.0.2. A p-value of < 0.05 was considered statistically significant. The experiments involving checkerboard assay, growth kinetic assay, pH determination in cultures, and biofilm metabolic evaluation were performed in triplicate with at least three independent cultures (n=3). Drug susceptibility testing with the co-culture system, co-incubation of microbial co-cultures with vaginal cells, hemolytic assay, and yeast-to-hyphal transition assessments were performed in triplicate with at least two independent cultures (n=2).

To standardize the co-culture conditions, multiple parameters were evaluated, including the selection of an optimum culture medium, initial densities of T. vaginalis, C. albicans and L. crispatus, incubation time, and cell viability assessment following incubation. Initially, four different media were tested to determine their ability to support the growth of all three microorganisms over a 24h incubation period. Only the presence or absence of growth was assessed ( Supplementary Table S1 ). MRS medium with 10% adult bovine serum and RPMI with 20% fetal bovine serum successfully supported microbial growth under these conditions. MRS medium was chosen for experiments without vaginal cells, while RPMI was used when vaginal cells were co-incubated with the microbial co-culture.

To standardize the initial cell density of each microorganism, the viability of T. vaginalis, L. crispatus, and C. albicans was compared between monoculture and co-culture conditions after 24 h of incubation. T. vaginalis viability was assessed using Trypan-blue exclusion assay and expressed as trophozoites/mL, while colony-forming units (CFU/mL) for C. albicans and L. crispatus were determined by colony quantification on selective agar ( Supplementary Table S2 ).

When ATCC 30236 T. vaginalis isolate was maintained at a constant density of 1.0 x 10⁶ trophozoites/mL (TV1) and co-cultured with L. crispatus at varying initial densities (LC1 - LC5; 5.53 x 10⁷ to5.53 x 10³ CFU/mL), trichomonads density decreased after 24 h. At the highest L. crispatus density (LC1, 5.53 x 10⁷ CFU/mL), T. vaginalis exhibited no growth. However, in co-cultures with LC2 (5.53 x 10⁶ CFU/mL), LC3 (5.53 x 10⁵ CFU/mL), LC4 (5.53 x 10⁴ CFU/mL), and LC5 (5.53 x 10³ CFU/mL), T. vaginalis density increased. At the two lowest L. crispatus densities (LC4 and LC5), T. vaginalis growth was comparable to the control (monoculture of T. vaginalis), with no significant difference observed.

When TV1 was maintained at a constant density and co-cultured with varying initial densities of C. albicans (CA1 - CA5; 3.33 x 10⁶ to 3.33 x 10² CFU/mL), only CA1 resulted in a significant change in trophozoite density after 24 h compared to T. vaginalis monoculture. The densities of C. albicans in co-cultures CA1, CA2, CA3, and CA4 were not significantly different from the C. albicans monoculture.

Similarly, when C. albicans was maintained at a constant initial density of 3.33 x 10⁴ CFU/mL (CA3) and co-cultured with L. crispatus, C. albicans viability after 24 h decreased in the presence of LC1 and LC2.The resulting CFU/mL counts were 3.75 ± 2.47 (x10⁵) CFU/mL and 8.25 ± 3.54 (x10⁵) CFU/mL, respectively, compared with the control (C. albicans monoculture) at 4.2 ± 1.41 (x10⁶) CFU/mL.

In triple co-culture, when TV1 (1.0 x 10⁶ trophozoites/mL) was maintained alongside CA3 (3.33 x 10⁴ CFU/mL) and LC2 (5.53 x 10⁶ CFU/mL), no significant difference in microbial densities was observed after 24 h compared to their respective monocultures. The recorded densities were T. vaginalis at 26.30 ± 1.34 (x10⁵) trophozoites/mL, C. albicans at 28.00 ± 6.36 (x10⁵) CFU/mL, and L. crispatus at 80.00 ± 2.83 (x10⁵) CFU/mL. Chequerboard assays, with LC2 maintained at 5.53 x 10⁶ CFU/mL confirmed no significant difference in the TV1 + CA3 + LC2 condition, yielding densities of 22.15 ± 2.15 (x10⁵) trophozoites/mL, 37.00 ± 3.18 (x10⁵) CFU/mL, and 80.00 ± 0.00 (x10⁵) CFU/mL, respectively. When CA3 (3.33 x 10⁴ CFU/ml) was held constant in the TV1 + CA3 + LC2 condition, T. vaginalis (24.17 ± 2.49 (x10⁵) trophozoites/mL) and L. crispatus (78.00 ± 2.12 (x10⁵) CFU/mL) densities remained unchanged, while C. albicans showed a significant difference to 35.00 ± 1.41 (x10⁵) CFU/mL. The TV1 + CA3 + LC4 condition also showed no significant differences compared to monocultures.

Based on these results, the TV1 + CA3 + LC2 combination was selected for further assays as it best mimicked an in vivo environment with higher L. crispatus density. Supplementary Table S2 lists all conditions and densities, while Table 1 summarizes monoculture and selected co-cultures conditions. Subsequent assays compared triple co-culture with dual co-cultures (TV1 + CA3, TV1 + LC2 or CA3 + LC2).

For the co-culture standardization using the TV-LACM6 fresh clinical isolate,T. vaginalis was maintained at 1x10⁶ trophozoites/mL, co-cultured with L. crispatus at its highest density (LC1; 5.53 x 10⁷ CFU/mL), and C. albicans (CA3; 3.33 x 10⁴ CFU/mL). Viability (%) was measured with monoculture controls set at 100% after 24h. T, vaginalis viability remained at 100% across all conditions remained at 100%. However, in co-culture with TV-LACM6, C. albicans and L. crispatus viabilities decreased to 58.55% and 85.89%, respectively. In the L. crispatus + C. albicans co-culture, L. crispatus viability was largely unaffected (97.36%), whereas C. albicans viability dropped significantly to 4.66%. In triple co-culture, L. crispatus viability significantly decreased to 83.12%, while C. albicans viability was 86.53% ( Table 2 ).

The growth kinetics assay revealed that T. vaginalis isolates reached their growth peak at 24h in both monoculture and co-culture with C. albicans and L. crispatus, after which trophozoite density declined ( Figures 1A, B ). In contrast, C. albicans and L. crispatus peaked at 48 h ( Figure 1C ). Therefore, a 24 h incubation period was chosen for co-culture to ensure all microorganisms were in their active growth phase.

The pH of culture medium was measured after 24 h in all monoculture and co-culture combinations of T. vaginalis isolates (ATCC30236 and TV-LACM6, TV1: 1.0 x 10⁶ trophozoites/mL), C. albicans (CA3, 3.33 x 10⁴ CFU/mL), and L. crispatus (LC1 and LC2, 5.53 x 10⁷ CFU/mL and 5.53 x 10⁶ CFU/mL). The medium alone and the C. albicans monoculture had a pH of 6.0. As expected, all combinations involving LC1, as well as the LC2 + CA3, had a pH of 4.0. However, when T. vaginalis isolates were present, the pH increased to 5.0 in the TV1 + LC2 and TV1 + CA3 + LC2 co-cultures ( Table 3 ). The pH values for co-cultures with ATCC30236 and TV-LACM6 isolates showed no significant differences.

As shown in Table 4 , the minimum inhibitory concentrations (MIC) of T. vaginalis isolates were determined using microdilution in microtiter plate wells, while the minimum bactericidal or fungicidal concentrations (MBC or MFC) for L. crispatus and C. albicans were assessed by sub-culturing on selective agar. Notably, monocultures exhibited higher MIC, MBC or MFC values for metronidazole (MTZ), fluconazole (FLZ), and streptomycin/penicillin (S/P) compared to co-cultures, highlighting the impact of microbial interactions on drug susceptibility.

Interestingly, treatment of T. vaginalis isolates with MTZ promoted the growth of L. crispatus at both densities (LC1 and LC2), resulting in CFU/mL counts of 40.25 ± 5.30 and 68.00 ± 1.41, respectively-significantly higher that the untreated co-culture values(30.50 ± 4.95 and 32.50 ± 2.12 CFU/mL). Conversely, when L. crispatus was treated with S/P, the growth of the TV-LACM6 T. vaginalis isolate increased, reaching19.20 ± 0.71 trophozoites/mL, compared to 16.00 ± 1.13 trophozoites/mL in the untreated co-culture. These findings emphasize the importance of the co-culture system in evaluating drug susceptibility, as microbial interactions can significantly influence treatment outcomes.

The cytolytic capacity of T. vaginalis can be measured by the release of the lactate dehydrogenase (LDH) enzyme from human vaginal epithelial cells (HMVII lineage). Figure 2A shows that co-incubation of the co-culture of ATCCT. vaginalis isolate (ATCC30236, at initial density 1.0 x 10⁶ trophozoites/mL), C. albicans (CA3, 3.33 x 10⁴ CFU/mL), and L. crispatus (LC2 5.53 x 10⁶ CFU/mL) with vaginal cells led to a significant increase in the LDH release compared to the co-incubation of ATC30236 alone with vaginal cells. On other hand, it is important to note that the co-incubation of C. albicans and two densities of L. crispatus (LC1, 5.53 x 10⁷ CFU/mL, and LC2, 5.53 x 10⁶ CFU/mL) showed no interference in LDH release ( Figures 2A, B ). The cytolytic capacity of TV-LACM6 fresh clinical isolate was more pronounced compared with ATCC30236. However, co-culture did not alter this effect, unlike what was observed with the standard isolate (ATCC).

The hemolytic activity of T. vaginalis was measured during co-incubation with erythrocytes and compared to its activity in co-culture with C. albicans and L. crispatus ( Figure 3 ). Similar to its interaction with vaginal cells, the ATCC isolate showed a significant increase in hemolysis when co-cultured with C. albicans and in triple co-culture with erythrocytes, compared to monoculture ( Figure 3A ). Notably, the hemolytic activity of the TV-LACM6 fresh clinical isolate was higher than that of ATCC30236 ( Figure 3B ). However, unlike the ATCC isolate, co-culture did not further enhance hemolysis in the clinical isolate.

The results of biofilm biomass and metabolic viability assessments are presented in Figure 4 . Biofilm biomass after 24 hours of incubation was evaluated in monocultures of C. albicans (CA3, initial density 3.3x10⁴ CFU/mL) and L. crispatus (LC2, initial density 5.53x10⁶ CFU/mL), which were set as the baseline (100%).

For C. albicans biofilm biomass, co-culture with the ATCC T. vaginalis isolate (ATCC30236, initial density 1.00x10⁶ trophozoites/mL) led to a 60% reduction, with biofilm decreasing to 40%).A more pronounced reduction (67%) was observed in the triple co-culture (ATCC30236, CA3, and LC2), resulting in a biofilm biomass of 33%).In the case of L. crispatus, co-culture with ATCC30236 led to a 96% reduction in total biofilm biomass (remaining biomass 4.0%), while the triple co-culture reduced biomass by 68% (remaining biomass 32%). Notably, the co-culture of CA3 and LC2 alone did not significantly impact biofilm biomass, which remained close to 100%, indicating that the presence of T. vaginalis played a crucial role in biofilm disruption ( Figure 4A ).

Biofilm metabolic viability was also assessed, expressed as a percentage relative to biofilm viability of the monocultures of CA3 and LC2, which were set as the baseline (100%). For C. albicans (CA3), co-culture with LC2 reduced viability by 21% (remaining viability 79%), When co-cultured with ATCC30236, metabolic viability was further reduced by 59% (remaining viability 41%), and in the triple co-culture (ATCC30236, CA3, and LC2), viability was inhibited by 89% (remaining viability 11%). In the case of L. crispatus (LC2), co-culture with ATCC30236 completely abolished biofilm viability (0%), while co-culture with CA3 resulted in a 33% reduction (remaining viability 77%). The triple co-culture significantly inhibited biofilm viability by 90% (remaining viability 10%) ( Figure 4A ).

A similar trend was observed when biofilm biomass and viability were evaluated using the fresh clinical T. vaginalis isolate (TV-LACM6). For C. albicans (CA3)biofilm biomass, co-culture with the TV-LACM6resulted in a 69% reduction (remaining biofilm biomass 31%). When co-cultured with LC1biofilmbiomass was reduced by 53% (remaining biomass 47%), while the co-culture (TV-LACM6,CA3, and LC1) resulted in a dramatic a 92% reduction (remaining biomass 8%).In the case of L. crispatus (LC1), co-culture with TV-LACM6 led to a 94% reduction in biofilm biomass (remaining biomass 6%), while co-culture with CA3 and LC1 resulted in a more moderate 16% reduction (remaining biomass 84%). The triple co-culture led to an 87% reduction in biofilm biomass (remaining biomass 13%) ( Figure 4B ).

Regarding metabolic biofilm viability co-culture with LC1 significantly increased C. albicans viability to 155%, suggesting a protective effect However, in the presence of TV-LACM6, biofilm viability was reduced by 53% (remaining viability 47%),and the triple co-culture (TV-LACM6, CA3, and LC1) resulted in a 66% reduction (remaining viability34%). L. crispatus (LC1) biofilm viability also increased in co-culture with CA3 (113%), while co-culture with TV-LACM6 significantly reduced biofilm viability by 93% (remaining viability 7%.The triple co-culture inhibited viability by 75% (remaining viability 25%).

Additionally, the yeast-to-hyphal transition of C. albicans (CA3) was quantified by microscopy, with yeast and hyphal forms expressed as percentages of the total observed forms (set as 100%) ( Figure 5 ). When CA3 was co-cultured with ATCC 30236, the yeast-to-hyphal transition rate was 84.1%, indicating an inhibition of 15.9%. The presence of L. crispatus (LC2) significantly reduced the transition rate to 4.41%, representing 95.6% inhibition. In the triple co-culture (ATCC30236, CA3, and LC2), the transition rate was 38.28%, reflecting an inhibition of 61.71% ( Figure 5A ).

A stronger inhibition effect was observed with the fresh clinical isolate (TV-LACM6). The yeast-to-hyphal transition rate in co-culture with TV-LACM6 was 30.09%, representing to 71.91% inhibition. Co-culture of LC1 with CA3 nearly abolished hyphal formation, with a transition rate of 0.63%, indicating 99.37% inhibition. The triple co-culture (TV-LACM6, CA3, and LC1) resulted in a transition rate of 29.23%, corresponding to 70.77% inhibition rate.

The morphology of T. vaginalis, C. albicans, and L. crispatus was analyzed using SEM in both monoculture and co-culture conditions. The T. vaginalis isolates ATCC30236 and TV-LACM6 were co-cultured with C. albicans (CA3) and L. crispatus (LC1 or LC2), under pre-selected conditions, and their morphology was evaluated after incubation. Figure 6A depicts the co-culture of the T. vaginalis ATCC30236 isolate (initial density, 1.00x10⁶ trophozoites/mL, marked with a red arrow), C albicans CA3 (3.3x10⁴ CFU/mL, orange arrow) and L. crispatus LC2 (5.53x10⁶ CFU/mL, white arrow). Similarly, Figure 6B , shows the co-culture of the fresh clinical isolate TV-LACM6 (1.00x10⁶ trophozoites/mL, blue arrow) with C albicans CA3 (orange arrow), and L. crispatus LC1 (5.53x10⁷ CFU/mL, white arrow).

The SEM micrographs revealed differences in trophozoite morphology between the isolates. In the triple co-culture on MRS medium, the ATCC30236 isolate exhibited a body length of 8.6 µm and width of 5.3 µm. In contrast, trophozoites of the fresh clinical isolate TV-LACM6 were smaller, measuring 6.1 µm in length and 4.33 µm in width. Regarding the bacterial and fungal counterparts, C. albicans ATCC 24433 was observed exclusively in yeast form, with an average length of 3.98 µm and width of 2.6 µm. Meanwhile, L. crispatus CCT 7595 measured 4.8 µm in length and 2.3 µm in width. These observations highlight morphological variations between the T. vaginalis isolates and confirm the distinct structural characteristics of C. albicans and L. crispatus under co-culture conditions.