The metabolic activity of planktonic and biofilm MRSA isolates was determined using a resazurin-based assay as previously described (82 (link)). An overnight culture of MRSA grown on a blood agar plate was used to inoculate 2 mL of 0.45% saline solution to 0.5 ± 0.1 McFarland turbidity standard (approximately 108 CFU/mL). For planktonic cultures, diluted cell suspensions (approximately 105 CFU/mL) were used to inoculate a 96-well polystyrene flat-bottom plate with 100 μL of an MHB/resazurin solution (Promega, Madison, WI, USA). The plates were incubated for 24 h at 37°C, and absorbance (570 nm) was recorded in 20-min periods for 1,200 min using a multidetection microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, USA).
For biofilm formation, 100 μL of diluted cells suspensions (approximately 105 CFU/mL) in MHB was transferred to a 96-well polystyrene flat-bottom plate. After 5 h at 37°C, the wells were rinsed with 0.45% saline solution, and 100 μL of an MHB/resazurin solution (Promega, Madison, WI, USA) was added. The plate was incubated for 20 additional hours at 37°C, and absorbance (570 nm) was recorded in 20-min periods for 1,200 min using a multidetection microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, USA).
For biofilm formation, 100 μL of diluted cells suspensions (approximately 105 CFU/mL) in MHB was transferred to a 96-well polystyrene flat-bottom plate. After 5 h at 37°C, the wells were rinsed with 0.45% saline solution, and 100 μL of an MHB/resazurin solution (Promega, Madison, WI, USA) was added. The plate was incubated for 20 additional hours at 37°C, and absorbance (570 nm) was recorded in 20-min periods for 1,200 min using a multidetection microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, USA).