Application suite 1

The effect of nicotine on the S. epidermidis biofilms was evaluated by LIVE/DEAD staining. Briefly, the biofilms were washed with PBS 3 times, and then stained with 1 μM of SYTO9, 1 μM of propidium iodide (PI), and 2.5 μg/ml Wheat Germ Agglutinin(WGA)-Alexa Fluor^® 350 conjugate (Thermo Fisher Scientific, United States) for 20 min. The stained cells and polysaccharide intercellular adhesin (PIA) were visualized by confocal laser-scanning microscopy (CLSM) (Leica TCS SP8 Confocal Laser Scanning Platform, Leica Microsystems, Germany) with a 63x 1.4-NA oil immersion objective. Three-dimensional biofilm images were created with IMARIS 7.0 software (Bitplane, United States). The red, green, and blue fluorescence intensity in each image was determined using Leica Application Suite 1.0 software (Leica Microsystem, Germany).

S. aureus biofilms were cultured in TSB with or without 2 mg/mL nicotine in glass-bottomed dishes, washed with three times with PBS, and then stained with LIVE/DEAD staining dye [1 μM of SYTO9 and 1 μM of propidium iodide (PI)] for 20 min. The biofilms were observed using a confocal laser-scanning microscope (Leica TCS SP8; Leica Microsystems, Germany) with a × 63 1.4-NA oil immersion objective. Fluorescence was quantified using Leica Application Suite 1.0 software (Leica Microsystem, Germany), and IMARIS 7.0 software (Bitplane, USA) was used to generate three-dimensional images of biofilms.

The effect of graRS knockout on the S. aureus biofilms was evaluated by LIVE/DEAD staining. The bacterial strains were cultivated in TSB supplemented with 1% glucose in glass-bottomed fluorodishes for 48 h. The biofilms were washed with PBS 3 times, stained with 1 μM of SYTO9 and 1 μM of propidium iodide (PI) for 20 min, and then visualized by confocal laser-scanning microscopy (CLSM) with a 63× 1.4-NA oil immersion objective (Leica TCS SP8 Confocal Laser Scanning Platform, Leica Microsystems, Germany). The three-dimensional biofilm images were generated with IMARIS 7.0 software (Bitplane, United States). The thicknesses of the biofilms and the fluorescence intensities were determined using Leica Application Suite 1.0 software (Leica Microsystem).