C. auris isolates from different origins were used: C. auris 467/15 (Venezuela) (Calvo et al., 2016), C. auris 136/18 (South Africa) (Magobo et al., 2014), C. auris 139/18 (Spain) (Ruiz-Gaitán et al., 2018), C. auris 138/18 (South Korea) (Lee et al., 2011), C. auris 140/18 (England) (Schelenz et al., 2016), and C. auris CBS 10913 (Japan) (Satoh et al., 2009). The reference strain C. albicans ATCC (American Type Culture Collection) 90028 and the clinical isolates of P. aeruginosa Pa14 (Rahme et al., 1997), Pa01 (Mikkelsen et al., 2011; Wettstadt et al., 2019), and Pak (Saliba et al., 2005) also were used. All strains were maintained in BHI medium (brain heart infusion; Kasvi, Brazil) supplemented with 10% glycerol at −80°C until use.

The spot-on-the-lawn antagonism method was performed according to Harris et al. (1989), with the modifications introduced by Peres-Emidio et al. (2022). Initially, 10 μL of P. aeruginosa Pa14, Pa01, and Pak (at 10⁶ colony-forming units [CFU]/mL), previously grown for 16 hours in BHI at 37°C, were spotted onto the center of Petri dishes containing BHI agar and incubated at 37°C for 18 hours.

The bacterial cells were then inactivated with 1 mL of chloroform vapor added to the lids of each plate. After 30 minutes of incubation at room temperature, the plates were left uncovered until the chloroform completely evaporated. Subsequently, 3 mL of semi-solid YPD medium (1% yeast extract, 2% peptone, 2% dextrose, and 0.75% agar), with 1×10⁵ CFU/mL of either C. albicans ATCC90028 or C. auris 467/15, was poured over the solid BHI medium containing the inactivated bacterial spot. After incubation for 48 hours at 37°C, the inhibition zones formed on the fungal cell lawn were visually measured. The C. auris and C. albicans cultures used in this assay were obtained from previous cultivation in YPD liquid medium for 24 hours.

Yeast inhibition was also evaluated in the co-cultivation of C. auris 467/15 with P. aeruginosa Pa14 in liquid medium (Peres-Emidio et al., 2022). Co-cultures and monocultures of C. auris and P. aeruginosa were prepared using different bacterial inocula (1×10⁵, 10⁶, and 10⁷ CFU/mL) and a fixed fungal inoculum of 1×10⁵ CFU/mL. The experiment was conducted in 1.5 mL tubes containing 1 mL of RPMI-1640 (Roswell Park Memorial Institute) medium (Gibco, USA) buffered with 34.5 g of MOPS (3-(N-morpholino) propanesulfonic acid) (Sigma-Aldrich, USA), with the pH adjusted to 7.0–7.2.

After incubation at 37°C for 24 hours, aliquots were plated on YPD medium with 0.2 mg/L of chloramphenicol. The plates were incubated at 37°C for 48 hours for CFU counting. The results were expressed in log CFU/mL, comparing fungal growth in monocultures and co-cultures (Peres-Emídio, 2020). The number of input cells in each case was similarly determined to ensure equivalent initial inocula of the bacterium and yeast.

As a control, the interaction between Candida albicans 90028 and P. aeruginosa Pa14 was included in all experiments, following the same protocol.

To broaden the findings obtained with the C. auris 467/15 strain, additional interaction assays in liquid medium were performed using C. auris isolates from diverse geographic origins: C. auris 136/18, 138/18, 139/18, 140/18, and CBS 10913. In these assays, a fungal inoculum of 1×10⁵ CFU/mL and a bacterial inoculum of 1×10⁶ CFU/mL of P. aeruginosa Pa14 were used.

A microbial kinetics experiment investigated the interaction between P. aeruginosa Pa14 and C. auris 467/15 over time. P. aeruginosa (1×10⁶ CFU/mL) was co-cultured with C. auris (1×10⁵ CFU/mL) in 1 mL of RPMI medium at 37°C—monoculture controls of each microorganism were also included. Samples were collected at 0, 4, 8, and 12 hours, and aliquots were plated on YPD agar supplemented with 0.2 mg/L chloramphenicol to determine yeast CFUs. To extend these findings, a subsequent experiment was conducted considering longer intervals: 0, 12, 24, 48, and 72 hours. At each time, aliquots were collected and plated to quantify bacterial CFUs on Cetrimide agar (Kasvi, Brazil) and yeast CFUs on YPD agar with 0.2 mg/L chloramphenicol.

Cell viability resulting from the interaction between C. auris 467/15 or C. albicans ATCC 90028 and P. aeruginosa Pa14 was assessed via fluorescence microscopy, following a modified protocol based on Sun et al. (2016). Co-cultures and monocultures were prepared in liquid RPMI medium as previously described, using inocula of 1×10⁶ CFU/mL for P. aeruginosa and 1×10⁵ CFU/mL for the fungi. After 72 hours of incubation at 35°C, the microtubes were centrifuged, the supernatant was discarded, and 300 μL of LIVE/DEAD (SYTO 9/PI) fluorescent staining solution—containing 5 μM SYTO 9 and 10 μg/mL propidium iodide (PI) (both from Invitrogen, USA), diluted in phosphate-buffered saline (PBS)—was added to the pellet. This solution stains viable cells green (SYTO 9) and dead cells red (PI).

Samples were incubated with the stain for 30 minutes at 35°C, protected from light. After that, the cells were washed twice with 300 μL of PBS, which was also used as the final resuspension volume. Finally, 20 μL of each sample was placed on a microscope slide, covered with a coverslip, and examined under a fluorescence microscope (EVOS FL Cell, USA).

Microscopic images from the co-culture and monoculture groups were processed using ImageJ software (https://imagej.net/ij/) to quantify live cells. Transmitted light (Trans) images, captured without a fluorescence filter, were converted to 8-bit, followed by thresholding and post-thresholding refinement steps, and used for automatic cell counting. RGB images, generated from the fluorescence emitted by cells labeled with IP and SYTO 9, were processed by selecting the red and green channels to generate a composite image, in which automatic cell labeling and manual cell counting were performed.

Results were expressed as total cell count, number of live cells, and percentage of live cells. Total cell counts from monocultures (C. auris 467/15 or C. albicans ATCC 90028) and co-cultures (C. auris 467/15 + P. aeruginosa Pa14/C. albicans ATCC 90028 + P. aeruginosa Pa14) were obtained from images captured under transmitted light (no filter). The number of live cells was determined by subtracting the number of dead cells (PI-labeled) from the total cell count in each condition. This value was cross-validated by comparison with the number of SYTO 9-labeled cells (green fluorescence). The percentage of live cells was calculated using the formula:

All counts were performed on five replicates derived from three independent experimental groups (biological triplicates) for both monocultures and co-cultures.

To investigate whether nutrient availability influences the bacterium-yeast interaction, co-cultures of P. aeruginosa Pa14 and C. auris 467/15, along with the respective controls, were performed in the following media: YPD, RPMI supplemented with 2% (w/v) glucose, 2-fold concentrated RPMI (RPMI 2x), and RPMI supplemented with FeSO₄ (at 30, 120, 240, 480, 960, and 1920 μM). The results were compared with those obtained using standard RPMI, following the previously described liquid co-culture protocol, using a 10:1 bacterium-to-yeast ratio.

P. aeruginosa Pa14 was cultured individually for 24 or 72 hours at 37°C. Following incubation, the cultures were centrifuged, and the supernatants were filtered through a 0.22 μm membrane to obtain the cell-free supernatant (CFS). The CFS was then mixed with RPMI in a 3:1 ratio (three parts CFS to one part 4-fold concentrated RPMI) and used to culture C. auris 467/15 at a final concentration of 1×10⁵ CFU/mL. As a control, C. auris was also cultured in a solution composed of three parts 0.85% NaCl (w/v) and one part 4-fold concentrated RPMI. After 24 hours of incubation at 37°C, yeast cells were plated on YPD agar, and CFUs were counted and compared across treatment groups.

P. aeruginosa Pa 14 (1×10⁶ cells/mL) was cultured for 24 hours in 10 mL of RPMI medium at 37°C. After incubation, the culture was centrifuged and filtered to obtain the CFS. The CFS and a control containing only RPMI were frozen and lyophilized for 48 hours, then reconstituted in 1 mL of RPMI. A portion of the reconstituted supernatant was supplemented with varying concentrations of ferrous sulfate (FeSO₄ at 30, 480, and 1920 μM). Subsequently, 100 μL of C. auris 467/15, adjusted to 1×10⁵ cells/mL, was added to 96-well plates with 100 μL of the reconstituted supernatant. The experimental groups comprised lyophilized cell-free supernatant (lCFS) of P. aeruginosa (lCFS_Pa) and lCFS of P. aeruginosa supplemented with FeSO₄ (lCFS_Pa + FeSO₄). Lyophilized RPMI and fresh RPMI were used as growth control groups.

The plates were incubated at 37°C, and visual assessments were conducted at 24, 48, and 72 hours. After 72 hours, aliquots from each experimental group were plated on YPD agar medium to quantify fungal viability.

The results were subjected to statistical analysis using PRISM 8.0 software (GraphPad Inc., San Diego, CA, USA) to assess significant differences among the experimental groups. One-way or two-way ANOVA was applied, followed by Tukey’s post-test or t-test at a 95% significance level. All experiments were performed independently at least three times.

The interaction between C. auris and P. aeruginosa was analyzed using the spot-on-the-lawn method and co-cultivation in a liquid medium. The spot-on-the-lawn assay revealed inhibition zones caused by products secreted by P. aeruginosa strains (PA14, PAO1, and PAK), demonstrating that C. auris (467/15) is inhibited by this bacterium ( Figure 1 ). The inhibition zones averaged 21.45 mm for strain PA14 (range = 20.4–21.5 mm), 21.31 mm for PAO1 (19.1–22.7 mm), and 21.19 mm for PAK (18.5–20.6 mm) ( Figure 1 ). A similar effect was observed in the interaction with C. albicans ( Figure 1 ).

Although no statistically significant difference was detected among the bacterial strains ( Figure 1 ), P. aeruginosa PA14 was selected for the subsequent experiments, as it has been widely used in fungal interaction studies (Peres-Emídio, 2020; Bastos et al., 2022).

Co-cultivation in a liquid medium was carried out to gain deeper insights into the phenomenon of antagonism. In the first step, varying concentrations of C. auris (10⁴, 10⁵, and 10⁶ CFU/mL) were co-cultured with a fixed concentration of P. aeruginosa (1×10⁶ CFU/mL) in RPMI liquid medium. As shown in Figure 2A , P. aeruginosa significantly inhibited yeast growth—by nearly 1 log cycle (10-fold) compared to C. auris grown in isolation—regardless of the fungal inoculum concentration. Notably, the inhibition was most pronounced at the lowest yeast inoculum (10⁴ CFU/mL). This result parallels the inhibitory effect observed for C. albicans when co-cultured with P. aeruginosa ( Figure 2B ).

Next, the yeast inoculum was kept constant (1×10⁵ CFU/mL) while the initial bacterial concentration varied (10⁵, 10⁶, and 10⁷ CFU/mL). The results showed that higher bacterial inocula led to more significant inhibition of C. auris ( Figure 2C ) and C. albicans ( Figure 2D ).

To investigate whether other C. auris isolates exhibit similar interaction behavior with P. aeruginosa, different C. auris strains were co-cultured in a liquid medium with the bacterium (1×10⁵ CFU/mL of yeast and 1×10⁶ CFU/mL of bacterium). Yeast concentrations were measured after 24 hours of co-cultivation.

Figure 3 shows that the growth of all six C. auris strains (467/15, 136/18, 138/18, 139/18, 140/18, and CBS 10913) was significantly inhibited by P. aeruginosa compared to monoculture. This indicates that P. aeruginosa exerts a consistent antagonistic effect against multiple C. auris strains.

After demonstrating that P. aeruginosa consistently inhibits different C. auris strains during co-cultivation, additional experiments were performed using C. auris 467/15. First, the kinetics of the interaction were investigated to establish the time point of maximum inhibition. Figures 4A, B show a substantial negative effect of P. aeruginosa on C. auris growth, which became significant between 8 and 12 hours of co-cultivation.

Further analysis revealed that the inhibition of C. auris by P. aeruginosa persisted steadily throughout the 72-hour evaluation period, with no statistically significant changes over time ( Figure 4C ). Interestingly, the fungal load in contact with the bacterium remained stable at approximately a 5-log cycle during the experiment ( Figure 4C ). A similar pattern was observed for C. albicans, which exhibited comparable inhibition under the same conditions ( Figure 4D ). Moreover, no significant differences were observed in bacterial concentrations between monoculture and co-culture conditions with either C. auris ( Figure 4E ) or C. albicans ( Figure 4F ), indicating that these yeasts were unable to inhibit the bacterium growth significantly.

As no significant changes in C. auris inhibition were observed over time during co-cultivation with P. aeruginosa from 12 to 72 hours ( Figure 4C ), we hypothesized that the bacterial effect might be fungistatic. To test this, C. auris and P. aeruginosa were cultured together and separately for 72 hours. The cells were then collected and stained with fluorescent markers to quantify total and live cells and calculate the live cell percentage.

A significant reduction in both total and live C. auris cell counts was observed in the co-culture with P. aeruginosa compared to monoculture ( Figures 5A, B ). Additionally, a slight decrease in the percentage of live cells was detected (monoculture mean = 99.58% vs. co-culture mean = 97.67%) ( Figures 5C ). Notably, the number of C. auris cells in co-culture reduced to 36% compared to monoculture; yet, 97% of these cells remained live after 72 hours of co-cultivation with P. aeruginosa. This slight reduction in viability, in contrast to the pronounced decline in total and live cell counts, suggests that P. aeruginosa exerts a fungistatic rather than fungicidal effect on C. auris, as the majority of yeast cells remain alive but fail to proliferate in the presence of the bacterium ( Figures 5A–C ).

A similar experiment conducted with C. albicans yielded comparable results. After 72 hours, a reduction in both total and viable cell counts was evident, along with a slight decrease in the percentage of viable cells, further supporting a fungistatic effect under the influence of P. aeruginosa ( Figures 5D–F ).

To evaluate whether different culture media and their compositions influence the interaction between C. auris and P. aeruginosa, we tested 2-fold concentrated RPMI, RPMI supplemented with 2% glucose, and liquid YPD, using standard RPMI as the reference condition. The inhibition of C. auris by P. aeruginosa remained consistent regardless of the medium used ( Figures 6A–C ). Moreover, the presence of additional nutrients, such as glucose in RPMI or the components of YPD, did not alter the inhibition pattern, showing no significant difference compared to the standard ( Figures 6A–C ).

The effect of iron supplementation on C. auris inhibition by P. aeruginosa was evaluated using RPMI medium supplemented with varying concentrations of FeSO₄. Iron significantly alleviated the inhibitory effect at all tested concentrations, with no further improvement observed beyond 30 μM ( Figure 7A ). It is important to note that the growth of C. auris in monoculture also was substantially enhanced by FeSO₄ supplementation (statistical analysis not shown).

To further confirm the impact of iron on the antagonistic effect of P. aeruginosa and to control for the growth enhancement caused by iron supplementation alone, we calculated the percentage of inhibition for each treatment by comparing C. auris growth in monoculture versus co-culture with P. aeruginosa in the presence and absence of FeSO₄ ( Figure 7B ). The results confirmed that iron concentrations between 30 and 480 μM significantly reduced growth inhibition, while higher concentrations did not produce additional benefits.

Overall, these findings indicate that Fe²⁺ supplementation partially restores C. auris growth in co-culture with P. aeruginosa compared to monoculture conditions.

Since P. aeruginosa inhibits the growth of C. auris even in the absence of viable bacterial cells (spot-on-the-lawn assay), it is likely that the bacterium produces and secretes molecules with fungistatic activity. To investigate whether this inhibition is mediated by secreted compounds, cell-free supernatant (CFS) was obtained after 24 hours of P. aeruginosa growth and used to culture C. auris and C. albicans. Unlike in co-culture conditions, the crude CFS did not inhibit yeast growth ( Figure 8A ). Similarly, the growth of P. aeruginosa was unaffected when cultured in crude CFS derived from C. albicans or C. auris cultures ( Figure 8A ).

The potential influence of bacterial culture duration before filtration was also evaluated. When P. aeruginosa was cultivated for an extended time (72 hours) before supernatant collection, the resulting CFS_Pa also failed to inhibit Candida growth ( Figure 8A ). To test whether the presence of the yeast is required to trigger the production of antagonistic metabolites, a co-culture was established for 24 hours, and the resulting CFS_{(Cau + Pa)} was subsequently used to culture C. auris for an additional 24 hours. Neither in this case was the growth of C. auris inhibited by the crude CFS_{(Cau + Pa)} ( Figure 8A ).

The inhibitory effect of P. aeruginosa CFS on C. auris was reassessed using a 10-fold concentrated supernatant obtained by lyophilization (lCFS_Pa). In 96-well plate assays, visual analysis confirmed that lCFS_Pa completely inhibited C. auris growth ( Figure 8B ). After 72 hours of incubation, lCFS_Pa effectively suppressed the yeast growth compared to RPMI alone, with similar inhibitory effects observed for C. albicans ( Figures 8B, C ).

To investigate the role of iron in modulating this interaction, lCFS_Pa was reconstituted in RPMI and supplemented with FeSO₄. At 30 μM, iron supplementation failed to reverse the inhibitory effect of lCFS_Pa. However, higher concentrations (480 and 1920 μM) significantly increased yeast growth compared to cultures with lCFS_Pa alone—although not to the same extent as observed in the RPMI control medium (without lCFS_Pa) ( Figures 8B, C ).

These findings confirm that P. aeruginosa secretes molecules with fungistatic activity against C. auris and C. albicans. Furthermore, this inhibitory effect is modulated by iron availability.