Lb agar

P. aeruginosa isolates were seeded on LB agar (Neogen, USA) and incubated at 37°C for 24 h. Then, one colony of each isolate was inoculated, in the presence and absence (control) of ½ MIC bio-AgNPs, to the bottom of twitching agar plates containing 1.0% tryptone (Acumedia, USA), 0.5% yeast extract (Bacto, Difco, USA), 1.0% sodium chloride (Synth, Brazil), and 1.0% agar (Acumedia, USA). Plates were inverted and incubated at 37°C for 24 h. Subsequently, the agar was carefully removed, and the motility zone was measured to the nearest millimeter after staining with 2% crystal violet (Laborclin, Brazil) for 2 h (Otton et al., 2017 (link)). As a negative control, each isolate was inoculated in tryptone soy agar (Difco, USA) under the same conditions.

Biofilm formation capacity was analyzed in 96-well polystyrene plates using the crystal violet method described by Ramos-Vivas et al. [21 (link)], with some modifications (Supplementary Table S1). Bacterial isolates were cultured on LB agar (Neogen, Lansing, MI, USA) at 37 °C for 24 h. Then, 180 µL of LB broth (Neogen, Lansing, MI, USA) and 20 µL of P. aeruginosa inoculum (initial concentration of 1.5 × 10⁶ CFU/mL) were added to each well and incubated at 37 °C for 24 h in the presence (1/2, 1/4, and 1/8 MIC) and absence (control) of bio-AgNPs. The supernatant of each well was discarded, and the cell layer was washed three times with phosphate buffer solution (PBS, pH 7.2), fixed with 250 µL of absolute methanol PA (Merck, Darmstadt, Germany) for 10 min, and stained with a 1.0% w/v aqueous solution of crystal violet (Merck, Darmstadt, Germany) for 15 min. Subsequently, the dye solution was discarded, and each well was washed three times with ultrapure water and treated with 250 µL of 33% v/v glacial acetic acid (Merck, Darmstadt, Germany). Assays were conducted in the presence (1/2, 1/4, and 1/8 MIC) and absence of bio-AgNPs. Absorbance readings were taken at 620 nm on a spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Experiments were repeated four times per isolate, and results are presented as mean and standard deviation.

Biofilm formation was analyzed on 96-well polystyrene microtiter plates by the modified crystal violet method described by Ramos-Vivas et al. (2019) (link). P. aeruginosa isolates were grown on LB agar (Neogen, USA) at 37°C for 24 h. Then, 180 µL of LB broth (Neogen, USA) and 20 µL of P. aeruginosa suspension (initial concentration of 1.5 × 10⁶ CFU/mL) were added to each well and incubated at 37°C for 24 h in the presence and absence (control) of ½ MIC bio-AgNPs. After incubation, the supernatants were discarded, and the wells were washed three times with phosphate-buffered saline (pH 7.2), fixed with 250 µL of methanol (Merck, Germany) for 10 min, and stained with a 1.0% crystal violet aqueous solution (Merck, Germany) for 15 min. Subsequently, the crystal violet was discarded and the wells washed three times with purified water. Adhered cells were resuspended in 250 µL of 33% (v/v) glacial acetic acid (Merck, Germany). Absorbance was read spectrophotometrically (Multiskan™ FC, Thermo Fisher Scientific, USA) at 620 nm.