Lb broth

P. aeruginosa strains were grown in LB broth (Neogen, Lansing, MI, USA) at 37 °C for 24 h in the presence (1/2, 1/4, and 1/8 MIC) or absence of bio-AgNPs. Then, 10 µL of supernatant from treated and untreated P. aeruginosa PAO1 and PA14 was added to a milk agar plate (pH 10.0) containing 1.0% w/v milk powder (Acumedia, Lansing, MI, USA), 0.1% w/v peptone (Acumedia, Lansing, MI, USA), 0.5% w/v NaCl (Synth, Diadema, SP, Brazil), and 2.0% w/v agar (Acumedia, Lansing, MI, USA) and was incubated at 37 °C for 24 h. Alkaline protease production was indicated by the formation of a clear halo around colonies. Halo diameters were measured and compared with those of the control [26 (link)].

For transformation, 1 µL of plasmid DNA (pNL4.3 Luc, VSV-g, and CMV) was added to 50 µL of super competent cells to a pre-chilled Luria-Bertani (LB) broth (Neogen, MI, USA) The transformation reaction was gently mixed and incubated on ice for 30 min. Thereafter, the reactions were placed on heat pulse for 45 s at 42 °C, followed by another incubation on ice for 2 min. Pre-heated super optimal broth with catabolite repression (S.O.C) medium (Thermo Fischer, Randburg, South Africa) at 42 °C was added to the transformation solution and incubated at 37 °C for 1 h, with shaking on a rotary evaporator at 230 rpm. Transformation reactions were plated on nutrient agar plates supplemented with 100 µg/mL ampicillin, then incubated at 37 °C for 16 h. After incubation, the colonies were transferred to the master plate using pipette tips. Then, the tips were dislodged into 15 mL of pre-heated LB broth supplemented with 100 µg/mL ampicillin and placed in a shaking incubator at 37 °C at 230 rpm.

P. aeruginosa isolates were grown in LB broth (Neogen, USA) for 24 h at 30°C. Then, swarming agar plates containing 1% glucose (Synth, Brazil), 0.5% peptone (Acumedia, USA), 0.2% yeast extract (Bacto, Difco, USA), and 0.5% agar (Acumedia, USA) were equilibrated to room temperature and inoculated at the center with 10 µL of a P. aeruginosa suspension containing 10⁸ colony-forming units (CFU)/mL in the presence and absence (control) of subinhibitory concentrations (½ MIC) of bio-AgNPs. Plates were incubated without inversion for 24 h at 30°C (Norizan et al., 2013 (link)).

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.

P. aeruginosa isolates were grown in LB broth (Neogen, Lansing, MI, USA) for 24 h at 30 °C. Ten microliters of a P. aeruginosa suspension containing 10⁸ CFU/mL in the presence (1/2, 1/4, and 1/8 MIC) or absence (control) of subinhibitory concentrations of bio-AgNPs were inoculated at the center of swarming agar plates containing 1% w/v glucose (Synth, Diadema, SP, Brazil), 0.5% w/v peptone (Acumedia, Lansing, MI, USA), 0.2% w/v yeast extract (BD, Sparks, MD, USA), and 0.5% w/v agar (BD, Sparks, MD, USA). Plates were incubated without inversion for 24 h at 30 °C [22 (link)].

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.

P. aeruginosa strains were cultured in the presence (1/2, 1/4, and 1/8 MIC) or absence of bio-AgNPs in LB broth (Neogen, Lansing, MI, USA) at 37 °C for 24 h. Then, 10 µL of inoculum was placed at the center of a cetyltrimethylammonium bromide (CTAB) agar plate containing 0.09% w/v monobasic potassium phosphate (Synth, Diadema, SP, Brazil), 0.11% w/v bibasic sodium phosphate (Synth, Diadema, SP, Brazil), 0.25% w/v sodium nitrate (Synth, Diadema, SP, Brazil), 0.01% w/v calcium chloride (Synth, Diadema, SP, Brazil), 0.04% w/v magnesium sulfate (Synth, Diadema, SP, Brazil), 0.2% w/v CTAB (Sigma–Aldrich, Steinheim am Albuch, Germany), 0.005% w/v methylene blue (Synth, Diadema, SP, Brazil), 0.5% w/v glucose (Synth, Diadema, SP, Brazil), and 2.0% w/v agar (Acumedia, Lansing, MI, USA). Plates were incubated at 37 °C for 48 h [25 (link)]. Rhamnolipid production was determined by measuring the halo of blue precipitate surrounding colonies.