Inhibiting Staphylococcus aureus Biofilms with 13-cis-Retinoic Acid

After forming S. aureus biofilms in 96-well plates in the presence or absence of 13-cis-retinoic acid (0, 2, 5, or 10 μg/mL) for 24 h at 37°C, planktonic cells were removed by washing three times with distilled water, and live biofilm cells were observed by the iRiS™ Digital Cell Imaging System (Logos Biosystems, Anyang, Korea). Color-coded 3D biofilm images were generated using ImageJ.¹Also, SEM was used to observe biofilm reduction by 13-cis-retinoic acid, as previously reported (Park et al., 2022 (link)). S. aureus ATCC 6538 cells (~10⁷ CFU/mL) were inoculated into 1 mL of fresh LB medium with or without 13-cis-retinoic acid (0, 2, 5, or 10 μg/mL) in a 96-well plate. A piece of nylon membrane (~ 0.16 cm²) was placed in each well, and S. aureus cells were cultured for 24 h at 37°C without agitation. Biofilms developed on the membrane were then fixed with a glutaraldehyde (2.5%) and formaldehyde (2%) for 24 h, post-fixed with OsO₄ (1%), and dehydrated with ethanol and isoamyl acetate (99%). After drying biofilms using critical-point dryer (HCP-2, Hitachi, Tokyo, Japan), biofilm cells were coated with Precision Etching Coating System (Gatan, Inc., Pleasanton, United States) and observed under a field emission scanning electron microscope S-4800 (Hitachi, Tokyo, Japan).