Syto9

Bacteria were cultured O/N in TSB + 1% glucose and diluted to an OD₆₀₀ = 0.05 (about 1 × 10⁷ CFU/mL) in the same medium. Bacterial suspension was added to the four-well Nunc Lab-Tek II Chambered Coverglass for two hours in TSB + 1% glucose, at 37 °C, in presence of different concentrations of C109 (0.625 or 1 μg/mL). The medium was removed, the biofilm was washed once in PBS to remove nonadherent cells, and fresh TSBG medium containing the same concentration of C109 was added. After an overnight incubation, the medium was removed, and biofilms were washed twice with PBS and stained with Syto 9 (Invitrogen) at a final concentration of 5 μM. A 63× oil immersion objective and a Leica DMi8 with 500- to 530-nm (green fluorescence representing Syto 9) emission filters were used to take five snapshots randomly at different positions in the confocal field of each chamber. The Z-slices were obtained every 0.3 microns. For visualization and processing of biofilm images, ImageJ was used. The thickness, biomass, roughness coefficient, and biofilm distribution were measured using the COMSTAT 2 software [48 (link)]. All confocal scanning laser microscopy experiments were performed three times, and standard deviations were measured.

To determine the effect of a combination of mAb and antibiotic treatment on biofilm formation, biofilm growth was induced in the presence or absence of 0.5 mg/ml 3H3, control 6A, or anti-CsgA. After 48 h, 30 μg/ml ampicillin was added. After an additional 24 h, biofilms were washed with PBS and stained with Syto9 and imaged using confocal microscopy as described above. Overnight cultures of wild-type S. Typhimurium were diluted 1:100 in LB No Salt broth with or without 0.5 mg/ml of 3H3 and grown for 72 h at 28 °C. After 72 h, 0.125 μg/ml ciprofloxacin or 12.5 μg/ml streptomycin was added to appropriate biofilms for an additional 24 h. Biofilms were washed four times to remove planktonic bacteria and then stained with 3 μl/ml Syto9 (Invitrogen) for 15 min and then washed four times with PBS. Syto9 was visualized at an excitation of 483 nm and emission of 503 nm at ×63 using a Leica SP5 Microscope equipped with a TCS confocal system. 3D surface plots were created using ImageJ Software.

B. cenocepacia K56-2 and the ΔBCAM0949 strains were cultured O/N in LB and diluted to an OD600 = 0.05 in the same medium. Bacterial suspension was added to the μSlide four chambered coverslip (Ibidi, Gräfelfing, Germany) for 4 h in LB, at 37 °C. The medium was removed and fresh LB medium was added. After overnight incubation, the medium was removed, and biofilms were washed twice with physiological solution and stained with Syto 9 (Invitrogen, Waltham, MA, USA) at a final concentration of 5 μM. A 63× oil immersion objective and a Leica (Wetzlar, Germany) DMi8 with 500 to 530 nm (green fluorescence representing Syto 9) emission filters were used to take five snapshots randomly at different positions in the confocal field of each chamber. The Z-slices were obtained every 0.3 microns. For visualization and processing of biofilm images, ImageJ was used. The thickness, biomass, roughness coefficient, and biofilm distribution were measured using the COMSTAT 2 software [51 (link)]. All confocal scanning laser microscopy experiments were performed three times, and standard deviations were measured.

Bacteria were cultured O/N in MH and diluted to an OD600 = 0.01 in the same medium. Bacteria were incubated in a four-well chambered coverslip μ-Slide (Ibidi) at 37 °C, in the presence of different concentrations of C109 (16, 64 and 256 mg/L). The compound C109 was dissolved in pure DMSO (≥99.9%), and the volume of C109 added was always 1/200 of the final volume, since this amount of DMSO was shown not to affect biofilm formation. After three hours of incubation the medium was removed, along with nonadherent cells, and fresh medium containing the same C109 concentrations, was added to the chambers. After over-night incubation, the medium was removed, and biofilms were washed with PBS 1X and stained with Syto 9 (Invitrogen, Waltham, MA, USA) at the final concentration of 5 μM. A 63X oil immersion objective Leica DMi8 with 500- to 530-nm (green fluorescence representing Syto 9) emission filters were used to take five snapshots randomly at different positions in the confocal field of each chamber. The Z-slices were obtained every 0.3 microns. For visualization and processing of biofilm images, ImageJ was used. The thickness, biomass, roughness coefficient, and biofilm distribution were measured using the COMSTAT 2 software [43 (link)]. All confocal scanning laser microscopy experiments were performed three times, and standard deviations were measured.

Biofilms of S. aureus SH1000, the isogenic Δpsm mutant, or E. coli UTI89 were grown on sterile glass circular coverslips or on medical mesh by diluting overnight cultures 1:100 in PNG media for S. aureus strains, or LB low salt media for UTI89, for 48 hours at 37°C (Schwartz et al., 2012 (link); Tursi et al., 2020 (link)). To visualize PSM in biofilms, coverslips were washed three times with sterile PBS and stained with 3 µg/mL Syto9 (ThermoFisher, S34854) for 10 minutes in the dark. Biofilms were then gently washed three times with sterile PBS and stained with 12.5 µM FSB (Millipore, 07602) or 10 ug/m Congo red (Sigma Aldrich, HT60-1KT) for an additional 10 minutes. Coverslips were placed upside down in 8-well Multi-test Slides (MP Biomedicals, 096040805E) with 3 μl Vectashield (Vector Labs, H-1000) between the coverslip and slide to prevent photo-bleaching. Syto9 was visualized with excitation at 483 nm and emission of 503 nm, FSB was visualized with excitation at 390 nm and emission of 511 nm, and Congo red was visualized with excitation at 561 nm and an emission of 650-750nm using a Leica TCS confocal imaging system at 63x magnification. Biofilm thickness was measured on Leica TCS imaging software.