Fig. 3: Effects of tizoxanide on other virulence factors of E. amylovora.

a Amylovoran production of E. amylovora TS3128 treated with tizoxanide. Amylovoran production was measured from the culture grown in MBMA medium containing 1% sorbitol and tizoxanide after a 48-h incubation at 28 °C. b Swarming motility of E. amylovora TS3128 treated with tizoxanide. Bacterial cells were spotted on SMM agar plates containing tizoxanide, and then, the diameter of bacterial spread was measured at 48 h post-inoculation. c Crystal violet-based biofilm assay. Biofilm from the cultures grown in LB medium containing tizoxanide for 48 h at 28 °C was quantified and visualized by crystal violet staining assay. d Confocal microscope images of the biofilm accumulation. Biofilm images were captured from E. amylovora strain containing pBAV1K-T5-gfp grown in LB medium containing tizoxanide for 6 h at 28 °C. The upper and lower panels were 2D and 3D biofilm accumulation, respectively. Scale bar, 40 µm. e Relative expression of genes involved in virulence factors of E. amylovora treated with different concentrations of tizoxanide. Total RNA was isolated from the E. amylovora TS3128 grown in induction medium for each virulence factor supplemented with tizoxanide. ns, not significantly different; *, P < 0.05; **, P < 0.01; and ***, P < 0.001 based on Student’s t-test.