Azithromycin Inhibits Biofilm Formation

Tissue culture plate assay was used to test the inhibitory effect of azithromycin on biofilm formation (Chen et al., 2017 (link)). A culture suspension of S. xylosus 700404 at the mid-exponential phase of growth was diluted with TSB to an optical density of 0.1 at 595 nm (OD₅₉₅). Next, 100 μL of this suspension and 100 μL of azithromycin were added to each well of a 96-well microplate, at final azithromycin concentrations of 1/2-MIC (0.25 μg/mL), 1/4-MIC (0.125 μg/mL), 1/8-MIC (0.0625 μg/mL), and 1/16-MIC (0.03125 μg/mL), respectively. In addition, control (with TSB alone) and negative control (with bacteria alone) were included after incubation at 37°C for 24 h without shaking. The supernatant was removed, the wells were rinsed three times with phosphate-buffered saline (PBS; pH 7.2), 200 μL of 99% methanol was added to the wells to fix the biofilms, and then, the plates were emptied after 15 min and stained for 5 min with 200 μL of 2% crystal violet per well (Ding et al., 2017 (link)). The wells were rinsed with PBS (pH 7.2), and the dye was resolubilized with 200 μL of 33% (v/v) glacial acetic acid per well (Ding et al., 2017 (link)). All wells were then measured using a Tecan GENios Plus microplate reader (Tecan, Austria) at 595 nm (Ding et al., 2017 (link)). The experiments were performed in triplicate.