Bacterial and yeast strains and their growth conditions

P. gingivalis wild-type strain W83 (ATCC BAA-308) purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and an isogenic gingipain-null mutant, ∆K∆RAB (lacking gingipains Kgp, RgpA, and RgpB), obtained as described previously [16], were cultured under anaerobic conditions (90% N₂, 5% CO₂, 5% H₂) at 37 °C on blood (5% (v/v) sheep blood) agar plates (BTL, Lodz, Poland) or in liquid tryptic soy broth (TSB) (30 g/l; Sigma-Aldrich, St Louis, MO, USA) with yeast extract (5 g/l; Bioshop, Burlington, Canada) supplemented with hemin (5 µg/ml), L-cysteine (50 µg/ml), and vitamin K (0.5 µg/ml) (all from Sigma-Aldrich), and additionally with tetracycline (1 µg/ml; Sigma-Aldrich) in the case of the ∆K∆RAB mutant. Bacteria from overnight liquid culture were centrifuged (4500 × g, 30 min) at 4 °C, washed three times, and resuspended in phosphate buffered saline, pH 7.4 (PBS; Biowest, Nuaillé, France). The optical density (OD) at 660 nm was measured with the use of a Shimadzu UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) to estimate the number of bacterial cells.

C. albicans strain 3147 (ATCC 10231) was purchased from American Type Culture Collection. Yeast cells were grown on YPD (1% yeast extract, 2% soybean peptone, and 2% glucose) agar (1.5%) plates (all components from Sigma-Aldrich) for 24 h at 30 °C and in liquid YPD medium for 18 h at 30 °C with shaking (170 rpm) using an orbital rotary shaker MaxQ 6000 (Thermo Fisher Scientific, Waltham, MA, USA) in aerobic conditions. Fungal cells from liquid cultures were harvested by centrifugation (3000 × g, 3 min), washed three times with PBS, and counted using OD measurements at 600 nm.

Single and mixed species biofilm formation

Biofilms were formed on the surface of flat-bottomed 96-well microplates (Corning, Glendale, AZ, USA). P. gingivalis and C. albicans cells were washed with PBS and suspended in RPMI 1640 medium buffered with 25 mM HEPES, pH 7.3 (Biowest), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Two independent suspensions were prepared: 2 × 10⁸ P. gingivalis cells /ml and 2 × 10⁷ C. albicans cells/ml. The wells of the plate were inoculated with 100 µl of each suspension for mixed biofilm formation or with 100 µl of one of the suspensions and the same volume of appropriate sterile medium (described above) to prepare single-species biofilms. The biofilms were incubated under aerobic conditions in the New Brunswick Galaxy 170R CO₂ incubator (Eppendorf, Hamburg, Germany) without shaking at 37 °C for 24 h.

For the purpose of standardizing the conditions for biofilm formation (number of microbial cells seeded in the wells, presence of FBS in the medium, and time of biofilm formation) with optimal protection of bacteria against aerobic conditions, evidenced by the highest gingipain production, a combination of 106 or 107 C. albicans cells/ml and 10⁸ P. gingivalis cells/ml, or each microorganism separately, were placed in RPMI 1640 with or without 10% FBS in the wells of a 96-well microplate. Then, biofilms were formed for 24 and 48 h at 37 °C under aerobic conditions.

Treatment of biofilms with antibiotics and antimycotics

Antibiotics and antifungal agents were added to the 24-hour-old mixed and single biofilms formed under aerobic conditions in a 5 µl volume to achieve final concentrations of levofloxacin (Sigma-Aldrich, 10 mg/ml stock in DMSO) at 0.1, 1, 10, or 50 µg/ml; meropenem (Sigma-Aldrich, 5 mg/ml stock in H₂O) at 0.1, 1, 10, or 50 µg/ml; metronidazole (Sigma-Aldrich, 10 mg/ml stock in H₂O) at 0.1, 1, 5, or 10 µg/ml; vancomycin (Sigma-Aldrich, 100 mg/ml stock in DMSO) at 0.1, 1, 5, 10, or 50 µg/ml; amphotericin B (Biowest, 250 µg/ml stock in 35% sodium deoxycholate and 10% phosphate buffer) at 0.25, 0.5, 1, or 2 µg/ml; caspofungin (Sigma-Aldrich, 10 mg/ml stock in H₂O) at 0.01, 0.1, 0.5, or 5 µg/ml; and fluconazole (Sigma-Aldrich, 20 mg/ml stock in DMSO) at 0.5, 5, or 50 µg/ml. Incubation of the biofilms with added drugs or biofilms without drugs (controls) was performed for 24 h under the same conditions as before treatment. The tests were performed at least three times in duplicate.

In another type of experiment, the combinations of antibiotic and antimycotic (levofloxacin with amphotericin B) were also added to the 24-hour-old biofilms formed under aerobic conditions. Both the administration of drugs and the further incubation of the biofilms were carried out as in the case of treatment of the biofilms with a single drug.

Gingipain activity measurement

Supernatants collected from 24-hour-old biofilms or from 48-hour-old biofilms untreated and treated with drugs were separated from the microbial cells by multiple centrifugation steps (once at 3000 × g for 5 min and three times at 6000 × g for 15 min). The proteolytic activity of gingipain R (Rgp) in each of the supernatants was determined by monitoring the hydrolysis of the chromogenic substrate benzoyl-l-arginine-p-nitroanilide (BApNA; Sigma-Aldrich), as previously described [29] with minor modifications. Briefly, 70 µl of assay buffer (200 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl₂, pH 7.6) supplemented with fresh 10 mM L-cysteine was mixed with 30 µl of supernatant in the wells of a microplate (Sarstedt, Nümbrecht, Germany). After 5-minute preincubation at 37 °C, 20 µl of BApNA (final concentration 0.5 or 4 mM) was added to the wells. The formation of p-nitroaniline was measured as an increase in the absorbance at 405 nm over a period of 1–2 h (with absorbance readings taken every minute) using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA).

Metabolic activity measurement

To determine the metabolic activity of microbial cells in biofilms, the 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2 H-tetrazolium-5-carboxanilide (XTT; Invitrogen, Waltham, MA, USA) reduction assay was used. The tests were performed as described previously [30] with some modifications. 24- or 48-hour-old single and mixed biofilms were washed once with PBS and 100 µl of RPMI 1640 without phenol red (Biowest) and 50 µl of a fresh mixture of XTT at a final concentration of 1 mg/ml and phenazine methosulfate (PMS; Sigma-Aldrich) at a final concentration of 5 µg/ml were added. After 40 min of incubation at 37 °C in the dark under aerobic conditions for mixed and monospecies C. albicans biofilms, 100 µl of supernatants were transferred to the wells of a new microplate (Sarstedt). The absorbance of the formazan product was measured at 450 nm using a Synergy H1 microplate reader (BioTek Instruments).

Microbial viability determination

To determine the viability of biofilm-forming microbial cells, a colony-forming unit (CFU) assay was used. The 48-hour-old biofilms were washed once, resuspended in PBS, scraped from the wells, homogenized by vortexing, and diluted ten thousand times in PBS. Then, 25 µl of the final suspensions were plated in quadruplicate on appropriate solid medium - YPD agar to determine the number of C. albicans cells, and blood agar to determine the number of P. gingivalis cells. They were cultivated aerobically at 30 °C for 24 h and anaerobically (in a GENbox jar anaerobic generator (bioMérieux, Craponne, France) at 37 °C for 5 days, respectively. After incubation, the numbers of colonies were counted. Each experiment was carried out at least three times in duplicate.

Permeabilization assay

After treatment with amphotericin B, the 48-hour-old mono- and dual-species biofilms were washed once with PBS, suspended in PBS containing 1 µM Sytox™ Orange Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA), and incubated for 5 min at room temperature in the dark. The permeabilization of the fungal membrane was visualized using an Olympus IX73 microscope (Olympus, Tokyo, Japan) equipped with a Hamamatsu Orca Spark camera (Hamamatsu, Hamamatsu City, Japan) and a UPLXAPO60XO lens (Olympus). All images were taken using the same parameters (exposure time, spectrum range), enabling the comparison of the fluorescence of the dye between different preparations, prepared in the form of Z-stacks (50 μm) and subjected to 3D deconvolution using CellSense software (Olympus).

Detection of metacaspase activation

The metacaspase activity was determined using a method previously described by Guevara-Lora et al. (2023) [31]. The 48-hour-old biofilms treated with amphotericin B were washed with PBS, suspended in 100 µl of staining solution containing CaspACE™ FITC-VAD-FMK In Situ Marker (Promega, Madison, WI, USA) at a concentration of 10 µM, and incubated for 1 h at room temperature in the dark under aerobic conditions. After this time, the biofilms were washed four times and then suspended in PBS. The green fluorescence of the marker bound to the active metacaspases was visualized using an Olympus IX73 microscope as described above.

Host cell culture and stimulation

Human bronchial epithelial BEAS-2B cells (ATCC CRL-9609) were purchased from ATCC. The cells were cultured in serum- and gentamycin-free BEGM medium (Lonza, Levallois-Perret, France) at 37 °C in an atmosphere of 5% CO2 and 95% humidity in bottles coated with 0.05 mg/ml of collagen (Sigma-Aldrich). Before each experiment, cells (4 × 104) were seeded in the collagen-covered wells of 96-well glass-like microplate (Cellvis, Sunnyvale, CA, USA) and cultured in BEGM for 24 h to 90% confluence. The medium was removed and 100 µl of the mixture of RPMI 1640 with 20% supernatant collected from 48-hour-old mixed biofilms (untreated or treated with levofloxacin (0.1–10 µg/ml) or levofloxacin and amphotericin B (0.5 µg), previously filtered with Ultrafree-CL Centrifugal Filters with a pore size of 0.22 μm (Merck, Darmstadt, Germany), was added to the cell monolayers. Then, BEAS-2B cells were stimulated for 24 h. Cells incubated in RPMI 1640 with 2% FBS, and in the same medium supplemented with levofloxacin (10 µg/ml) or combination of levofloxacin (10 µg/ml) and amphotericin B (0.5 µg/ml), served as controls.

Apoptosis and host cell death analysis

After a 24-hour incubation with sterile biofilm supernatants, BEAS-2B cells were washed once with PBS and then suspended in 50 mM HEPES buffer with 700 mM NaCl and 12.5 mM CaCl2 containing Annexin V Alexa Fluor® 488 (Invitrogen, Waltham, MA) diluted twenty-fold. Cells were stained at room temperature for 15 min, then washed three times with PBS and incubated for 10 min at room temperature in RPMI 1640 medium without phenol red (Biowest) with added 1 µM Sytox™ Orange Nucleic Acid Stain. Cell visualization was performed using an Olympus IX73 microscope and the obtained images were processed with Olympus CellSens Dimension 3.1 imaging software.

IL-8 detection assay

To measure IL-8 production levels, BEAS-2B cells were stimulated as described above, except for the concentration of biofilm-derived sterile supernatants, which was 2.5%. After 24-hour incubation, supernatants collected from above BEAS-2B cells were centrifuged to remove accidentally detached cells (once for 5 min at 3000 rpm; twice for 10 min at 7000 rpm, each time with the sediment discarded). The level of IL-8 produced by human cells was determined using the Human IL-8 ELISA Set (BD Biosciences, San Diego, CA, USA OptEIA™) strictly according to the manufacturer’s instructions.

Statistical analysis

Statistical analyses were performed with the GraphPad Prism 8 software (GraphPad Software, San Diego, CA, United States). To evaluate the significance between groups, one-way ANOVA with Dunnett’s multiple comparison post hoc test was used. The results were considered statistically significant at value of p < 0.05: *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001.