Antimicrobial Liposomal Biofilm Inhibition

The liposomal formulations were next tested for their inhibitory effects on biofilm viability, as previously described (Graziano et al., 2015 (link)) (39). Cellulose acetate membranes (25 mm diameter, 0.2 µM pores) (Sartorius Stedim GmbH, Guxhagen, Hessen, Germany) were used as substrates for S. aureus biofilm formation. The membranes were placed in 6-well plates containing BHI medium supplemented with 1% glucose and bacterial suspension (approximately 1 × 10⁶ CFU/ml in each well). The plates were incubated at 37°C for 24 h. Then the membranes were transferred to new plates containing fresh BHI plus 1% glucose, and biofilms were treated with the formulations at 1 × MIC, 10 × MIC, and 50 × MIC for 24 h. Treated biofilm-coated membranes were gently washed (three times) through immersion into 5 ml of 0.9% NaCl. Then, the membranes were transferred to other tubes containing freshly 5 ml of 0.9% NaCl and then sonicated with six pulses of 9.9 s, 5 s time-interval, 5% amplitude (VibraCell 400W, Sonics & Materials Inc., Newtown, CT, USA) and vortexed at 3,800 rpm for 30 s. Ten-microliter aliquots were collected from each tube, serially diluted, and plated for CFUs onto TSA. The plates were incubated at 37°C for 24 h.