Sterile glass coverslips

Sterile glass coverslips (Hampton Research, Aliso Viejo, CA, United States) were plunged into overnight A. baumannii Ts 50-16 culture in tryptic soy broth (TSB) medium in Petri dishes and incubated at 37°C for 24h without shaking for biofilm formation. Then, slides were washed with distilled water three times and air-dried. Three slides were left intact, three were submerged into 20mM Tris–HCl (pH=7.5) control buffer, other slides were submerged into 100μg/ml LysAm24, LysAp22, LysECD7, or LysSi3 solutions, with three slides for each solution, respectively, for 2h at RT. Afterward, all slides were again washed with distilled water three times and air-dried. One slide from each group was fixed in 10% formalin vapors for 24h and analyzed using scanning electron microscopy after coating with a gold layer, as described above. Another slide from each group was imprinted on LB agar to assess bacterial viability. The remaining slides were stained with 0.1% aqueous solution of crystal violet for 20min at RT, washed three times with water, and dried. These slides were analyzed using an Axiostar Plus Transmitted Light Microscope (Zeiss AG, Jena, Germany) at ×400 magnification.

For microscopy, dual-species biofilms (Pseudomonas-Staphylococcus) were preformed on a glass. Sterile glass coverslips (Hampton Research, Aliso Viejo, CA, USA) were plunged in Petri dishes containing the bacterial cultures mixture 1:1 (v/v) in TSB with approximately 10⁶ CFU/mL of each strain. The dishes were incubated at 37 °C for 48 h without shaking. Slides were carefully washed with sterile water, and then, 10 μL of SiL-gel or the vehicle gel (Alg-base) was dropped onto the slides, and they were incubated for 2 h at room temperature. Afterward, the slides were again carefully washed with sterile water two times.
Air-dried slides were stained with a 0.1% crystal violet solution for 15 min at RT, rinsed with water, and air-dried for bright-field microscopy. Other slides were stained with the DAPI Nucleic Acid Stain (Molecular Probes, Invitrogen, Eugene, OR, USA), the FilmTracer™ SYPRO^® Ruby Biofilm Matrix Stain (Molecular Probes, Invitrogen, Eugene, OR, USA), and the FilmTracer™ LIVE/DEAD^® Biofilm Viability Kit (Molecular Probes, Invitrogen, Eugene, OR, USA), according to the manufacturers’ instructions for dark-field fluorescent microscopy. All slides were imaged with the Axiostar Plus Transmitted Light Microscope (Zeiss AG, Jena, Germany) at ×400 and ×630 magnification. Microphotographs were proceeded with the ZEN 3.0 (blue edition) software (Zeiss AG, Jena, Germany).